Methods for diagnosing and treating hemostatic disorders by modulating P-selectin activity

ABSTRACT

The present invention identifies P-selectin as a modulator of hemostasis. Accordingly, the present invention relates to methods for the identification and use of modulators of P-selectin activity as modulators of hemostasis. The invention also relates to methods and compositions for the diagnosis and treatment of hemostatic disorders, including, but not limited to, hemorrhagic disorders and thrombotic disorders. The present invention describes methods for the diagnostic evaluation and prognosis of various hemostatic conditions, and for the identification of subjects exhibiting a predisposition to such conditions. In addition, the present invention provides methods for the diagnostic monitoring of patients undergoing clinical evaluation for the treatment of a hemostatic or vascular disorders, and for monitoring the efficacy of compounds in clinical trials.

RELATED APPLICATIONS

[0001] This application claims the benefit of prior-filed provisionalpatent application Ser. No. 60/205,734, filed May 19, 2000, entitled“Methods For Diagnosing and Treating Hemostatic Disorders By ModulatingP-Selectin Activity.” The entire content of the above-referencedapplication is incorporated herein by this reference.

BACKGROUND OF THE INVENTION

[0002] The ability of cells to adhere to one another plays a criticalrole in development, normal physiology, and disease processes. Thisability is mediated by adhesion molecules, generally glycoproteins,expressed on the cell surface. Several important classes of adhesionmolecules include the integrins, the selecting, and members of theimmunoglobulin (Ig) superfamily. Selectins play a central role inmediating leukocyte adhesion to activated endothelium and platelets.

[0003] Blood clotting, along with inflammation and tissue repair, arehost defense mechanisms which function in parallel to preserve theintegrity of the vascular system after tissue injury. In response totissue injury, platelets, endothelial cells and leukocytes are essentialfor the formation of a platelet plug, deposition of leukocytes ininjured tissue, initiation of inflammation, and wound healing.

[0004] P-selectin, also known as CD62, granule membrane protein-140(GMP-140), and platelet activation-dependent granule external membraneprotein (PADGEM), is an integral membrane glycoprotein that is expressedon vascular endothelial cells and platelets, and is involved in therecognition of various circulating cells. The P-selectin molecule has anN-terminal lectin domain, a region with homology to epidermal growthfactor, a region with homology to complement regulatory proteins, atransmembrane domain, and a short cytoplasmic tail. The P-selectinligand includes the Lex carbohydrate structure, sialic acid, and thePSGL-1 protein (U.S. Pat. No. 5,843,707).

[0005] P-selectin is constitutively stored in secretory granules (e.g.,a-granules and Weibel-Palade bodies) and is translocated to the surfaceof platelets and endothelial cells in response to a variety of stimuli,including cell activation, where it mediates platelet-leukocyte andendothelium-leukocyte interactions. The cell surface expression ofP-selectin is tightly regulated, and P-selectin is rapidly shed from thecell surface upon platelet activation, appearing as a soluble fragmentin the plasma (Berger, G. et al. Blood (1998) 92:4446-4452). SolubleP-selectin may also result from an alternatively spliced isoform ofP-selectin lacking the transmembrane domain (Ishiwata, N. et al. J. BiolChem (1994) 269:23708). The plasma of healthy humans and mice containslittle soluble P-selectin, as detected by ELISA, and an increase inplasma P-selectin concentration may indicate in vivo activation ofand/or damage to platelets and endothelial cells.

[0006] In addition to its role in leukocyte rolling and extravasation ininflammation, P-selectin mediates platelet-leukocyte adhesion withinthrombi, and increases tissue factor expression on monocytes, therebypromoting fibrin deposition by leukocytes and thrombogenesis (Palabrica,T. et al. Nature (1992) 359:848-851; Celi, A. et al. Proc Natl Acad SciUSA (1994) 91:8767-8771).

SUMMARY OF THE INVENTION

[0007] The present invention provides methods and compositions for theregulation of hemostatic and thrombotic processes using modulators ofP-selectin activity (e.g., inducers and inhibitors of P-selectinactivity), as well as for the diagnosis and treatment of hemostaticdisorders.

[0008] In one aspect, the invention provides methods for inducinghemostasis in a subject, comprising administering an inducer ofP-selectin activity to the subject. In one embodiment, the inducer ofP-selectin activity increases the level of circulating solubleP-selectin in the subject. The inducer of P-selectin activity mayincrease the level of soluble P-selectin polypeptide by increasing theproteolytic cleavage of P-selectin from the cell surface, or byincreasing P-selectin gene expression. In another embodiment, theinducer of P-selectin activity binds to a P-selectin ligand or receptor(e.g., PSGL-1) and mimics the activity of a P-selectin polypeptide,e.g., a soluble P-selectin polypeptide.

[0009] In an exemplary embodiment, the invention provides methods forinducing hemostasis in a subject, comprising administering solubleP-selectin polypeptide to the subject. In another embodiment, anisolated nucleic acid molecule comprising a nucleotide sequence whichencodes a soluble P-selectin polypeptide is administered to the subjectto induce hemostasis. In a further embodiment, hemostasis is induced ina subject by administering a recombinant cell expressing solubleP-selectin polypeptide.

[0010] In another aspect, the invention provides methods for treating orpreventing a disorder associated with hypocoagulation, e.g., ahemorrhagic disorder, in a subject, comprising administering to thesubject an inducer of P-selectin activity. In one embodiment, a solubleP-selectin polypeptide is administered to a subject to treat or preventa disorder associated with hypocoagulation.

[0011] In a further aspect, the invention provides methods for treatinga vasculature-associated disease in a subject, comprising administeringto the subject an inducer of P-selectin activity. In a preferredembodiment, a soluble P-selectin polypeptide is administered to asubject to treat or prevent a vasculature-associated disease. In oneembodiment, the vasculature-associated disease is a tumor. In anotherembodiment, the subject is further treated with a molecule effective toinduce a procoagulant state in tumor associated vasculature, e.g., amolecule comprising a first binding region that binds to a component ofa tumor cell or tumor associated vasculature operatively linked to acoagulation factor or a second binding region that binds to acoagulation factor.

[0012] Another aspect of the invention provides methods for reducinghemostasis in a subject, comprising administering to the subject aninhibitor of P-selectin activity. In one embodiment, the inhibitor ofP-selectin activity decreases the level of soluble P-selectin in plasmaof the subject. The inhibitor of P-selectin activity may decrease thelevel of the soluble P-selectin polypeptide by decreasing theproteolytic cleavage of P-selectin from the cell surface, or decreasingP-selectin gene expression. In another embodiment, the inhibitor ofP-selectin activity is an anti-P-selectin antibody. In yet anotherembodiment, the inhibitor of P-selectin activity is a recombinantsoluble PSGL-1 polypeptide. In a further embodiment, the inventionprovides a method for reducing hemostasis in a subject, comprisingadministering an isolated nucleic acid molecule comprising a nucleotidesequence which is antisense to a nucleotide sequence which encodes aP-selectin polypeptide, e.g., a soluble P-selectin polypeptide.

[0013] In another aspect, the invention provides methods for treating orpreventing a thrombotic disorder in a subject, comprising administeringto the subject an inhibitor of P-selectin activity. Thrombotic disordersthat may be treated or prevented using the methods of the inventioninclude arteriosclerosis, deep vein thrombosis, angina, e.g., unstableangina, and restenosis following medical intervention.

[0014] In a further aspect, the invention provides methods formodulating hemostatic potential in a subject, comprising modulatingP-selectin activity in the subject. In one embodiment, a modulator(e.g., an inducer or inhibitor) of P-selectin activity is administeredto a subject to modulate hemostatic potential. A modulator of solubleP-selectin activity may act by regulating the level of solubleP-selectin in the plasma of the subject.

[0015] Another aspect of the invention provides a method for diagnosinga procoagulant state in a subject, comprising determining an increasedlevel of P-selectin activity in a biological sample of the subject. Inone embodiment, the level of soluble P-selectin in a test sample ofblood or plasma from a subject is compared to the level of solubleP-selectin in a control blood or plasma sample from a subject withnormal hemostatic activity, wherein an increased level of solubleP-selectin in the test sample as compared to the control sample is anindication of a procoagulant state in the subject.

[0016] In another aspect, the invention provides a method foridentifying a subject having a thrombotic disorder, or at risk fordeveloping a thrombotic disorder, comprising determining an increasedP-selectin activity in a biological sample of the subject. In oneembodiment, a sample of blood or plasma obtained from a subject iscontacted with a P-selectin binding substance, and the detection ofincreased levels of soluble P-selectin polypeptide in the sampleidentifies a subject having a thrombotic disorder, or at risk fordeveloping a thrombotic disorder.

[0017] Another aspect of the invention provides a method for identifyinga compound capable of modulating hemostasis, comprising assaying theability of the test compound to modulate a P-selectin activity. In oneembodiment, the P-selectin activity is the expression of solubleP-selectin.

[0018] In a further aspect, the invention provides compositions formodulating hemostasis comprising at least one modulator of P-selectinactivity.

[0019] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is a photograph of en face examination of the thromboticdeposits in wild-type mice (WT), P-selectin deficient mice (PKO), andΔCT mice formed after a 2 minute non-anticoagulated blood perfusion(blood flow, left to right). The white arrow indicates platelet richthrombus; the black arrow indicates fibrin tail formed distally to theplatelet thrombus.

[0021]FIG. 2 shows fibrin formation in a perfusion chamber ofnon-anticoagulated blood from wild type mice (WT), P-selectin deficientmice (P-sel −/−), and ΔCT mice.

[0022]FIG. 3 shows macroscopic and microscopic grading of hemorrhagiclesions formed in a local Shwartzman reaction in wild type mice (WT)that were either untreated, perfused with human IgG1, or perfused withsoluble P-selectin-Ig (s-P-sel), and ΔCT mice.

[0023]FIG. 4 shows fibrin deposition in a local Shwartzman reaction inwild type (WT) mice that were perfused with either human IgG1 or solubleP-selectin-Ig (P-sel).

[0024]FIGS. 5A and B show the plasma clotting time of wild type mice(WT), P-selectin deficient mice (P-sel −/−), and ΔCT mice that wereeither untreated or perfused with recombinant PSGL-1 or recombinantsoluble P-selectin.

[0025]FIG. 6 shows the levels of microparticles in the circulation ofwild type mice (WT) that were either untreated, perfused with humanIgG1, or perfused with soluble P-selectin-Ig (s-P-sel), and ΔCT mice.

[0026]FIG. 7 shows the number of microparticles expressing tissue factorin wild type (WT) and ΔCT mice.

[0027]FIG. 8 shows the increased generation of procoagulantmicroparticles in the circulation of von Willebrand factor deficientmice (vWF −/−) that were perfused with soluble P-selectin-Ig(sP-sel-Ig).

[0028]FIG. 9 shows the prothrombin clotting time of wild type mice (WT),and von Willebrand factor deficient mice (vWF −/−) that were eitheruntreated, perfused with human IgG1, or perfused with solubleP-selectin-Ig (sPselIg).

[0029]FIG. 10 shows the bleeding time in hemophilia A mice treated witheither human IgG1 or soluble P-selectin-Ig (P-sel-Ig).

[0030]FIG. 11A shows the reduction in the number of microparticles aftertreatment of ΔCT mice with soluble PSGL-Ig as compared to control humanIg (*=p<0.05).

[0031]FIG. 11B shows the increase in clotting time after treatment ofΔCT mice with soluble PSGL-Ig as compared to control human Ig(*=p<0.05).

[0032]FIG. 12A shows the generation of procoagulant microparticles inhuman blood after incubation with either human IgG or solubleP-selectin-Ig (P-sel-Ig). After 6 hrs. incubation with solubleP-selectin-Ig, the numbers of microparticles significantly increased by30% (*=p<0.04).

[0033]FIG. 12B shows the generation of tissue factor positivemicroparticles in human blood after incubation with either human IgG orsoluble P-selectin-Ig (P-sel-Ig). The number of tissue factor positiveevens was significantly increased at 6 hours by incubation withP-selectin Ig, 30% (*=p<0.05).

[0034]FIG. 13A shows the clotting time of human whole blood afterincubation with human IgG or soluble P-selectin-Ig (P-sel-Ig). Theclotting time of whole blood incubated with soluble P-selectin-Ig wasshortened by about 20% after 2 hours (*=p<0.02) and by 60% after 8 hoursof incubation (**=p<0.004).

[0035]FIG. 13B shows the clotting time of human plasma after incubationwith human IgG or soluble P-selectin-Ig (P-sel-Ig). The plasma clottingtime of the soluble P-selectin treated blood was shortened by 25% after6 hours of incubation and by 40% after 8 hours (**p<0.004).

[0036]FIG. 14A shows activated partial thromboplastin time (APTT) infactor VIII −/− mice (hemophilia A mice) treated with control Ig orsoluble P-selectin-Ig.

[0037]FIG. 14B shows plasma clotting time in factor VIII −/− mice(hemophilia A mice) treated with control Ig or soluble P-selectin-Ig.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention provides modulators (e.g., inducers,inhibitors) of P-selectin activity as therapeutic and diagnostic agentsfor the regulation of hemostasis. The present invention is based on thediscovery that soluble P-selectin induces a procoagulant state in amammal, for example a mouse or a human, (e.g., by increasing the numbersof microparticles containing tissue factor in the blood, reducingbleeding time, and/or reducing clotting time).

[0039] As used herein, the term “modulator of P-selectin activity”includes a compound or agent that is capable of modulating or regulatingat least one P-selectin activity, as described herein. In a preferredembodiment, a modulator of P-selectin activity modulates the expressionof soluble P-selectin. A modulator of P-selectin activity can be aninducer of P-selectin activity or an inhibitor of P-selectin activity.As used herein, an “inducer of P-selectin activity” stimulates,enhances, and/or mimics a P-selectin activity. As used herein, an“inhibitor of P-selectin activity” reduces, blocks or antagonizes aP-selectin activity.

[0040] As used interchangeably herein, a “P-selectin activity”,“biological activity of P-selectin” or “functional activity ofP-selectin” refers to an activity exerted by a P-selectin polypeptide ornucleic acid molecule on a P-selectin responsive cell (e.g., ahematopoietic cell or lymphocyte) or tissue, or on a P-selectin ligandor receptor, as determined in vitro and in vivo, according to standardtechniques. In an exemplary embodiment, a P-selectin activity is theability to modulate hemostasis. In one embodiment, a P-selectin activityis a procoagulant activity. In another embodiment, a P-selectin activityis the ability to increase the number of microparticles containingtissue factor. In yet another embodiment, a P-selectin activity is theability to bind a P-selectin ligand, e.g., PSGL-1.

[0041] Accordingly, the invention provides a method for regulatinghemostasis in a subject, at least in part, by increasing or decreasingP-selectin activity in the subject (e.g., by increasing or decreasinglevels of circulating soluble P-selectin). As used interchangeablyherein, the terms “hemostasis”, “hemostatic activity”, or “hemostaticpotential” refer to the control of bleeding, including the physiologicalproperties of vasoconstriction and coagulation. Blood coagulationassists in maintaining the integrity of mammalian circulation afterinjury, inflammation, disease, congenital defect, dysfunction or otherdisruption. After initiation of clotting, blood coagulation proceedsthrough the sequential activation of certain plasma proenzymes to theirenzyme forms (see, for example, Coleman, R. W. et al. (eds.) Hemostasisand Thrombosis, Second Edition, (1987)). These plasma glycoproteins,including Factor XII, Factor XI, Factor IX, Factor X, Factor VII, andprothrombin, are zymogens of serine proteases. Most of these bloodclotting enzymes are effective on a physiological scale only whenassembled in complexes on membrane surfaces with protein cofactors suchas Factor VIII and Factor V. Other blood factors modulate and localizeclot formation, or dissolve blood clots. Activated protein C is aspecific enzyme that inactivates procoagulant components. Calcium ionsare involved in many of the component reactions. Blood coagulationfollows either the intrinsic pathway, where all of the proteincomponents are present in blood, or the extrinsic pathway, where thecell-membrane protein tissue factor plays a critical role. Clotformation occurs when fibrinogen is cleaved by thrombin to form fibrin.Blood clots are composed of activated platelets and fibrin.

[0042] As used herein, the term “procoagulant state” includesphysiological conditions that are conducive to and/or promote bloodclotting, hemostasis, and/or thrombosis. Hemostatic potential, e.g., thepotential for blood coagulation under the appropriate physiologicalconditions, or hemostatic activity can be assessed using wellestablished laboratory tests including prothrombin time (PT), activatedpartial thromboplastin time (APTT), bleeding time, and thrombin time. Asused interchangeably herein, “modulating or modulation of hemostasis”and “regulating or regulation of hemostasis” includes the induction(e.g., stimulation, increase) of hemostasis, as well as the inhibition(e.g., reduction, decrease) of hemostasis.

[0043] In one aspect of the invention, hemostasis is induced in asubject by administering an inducer of P-selectin activity. In anexemplary embodiment, an inducer of P-selectin activity increases theplasma level of soluble P-selectin polypeptide. In this respect, aninducer of P-selectin activity may act to stimulate the translocation ofP-selectin from a cellular storage pool to the cell surface, or toincrease the proteolytic cleavage and release of soluble P-selectin fromthe surface of a cell expressing P-selectin, e.g., an endothelial cellor a platelet. In another embodiment, an inducer of P-selectin activityincreases P-selectin gene expression by stimulating either genetranscription or translation. In a preferred embodiment, an inducer ofP-selectin activity will preferentially stimulate the expression of analternatively spliced isoform of the P-selectin gene encoding a solubleP-selectin polypeptide lacking the transmembrane domain. In yet anotherembodiment, an inducer of P-selectin activity binds to a P-selectinligand or receptor (e.g., PSGL-1) and mimics the activity of aP-selectin polypeptide on a P-selectin responsive cell. The inducer ofP-selectin activity can thereby elicit a biological response ofP-selectin, e.g., the release of microparticles containing tissuefactor. Accordingly, in one embodiment, an inducer of P-selectinactivity is an antibody, e.g., an anti-PSGL-1 antibody.

[0044] In another embodiment of the invention, a soluble P-selectinpolypeptide is administered to a subject to induce hemostasis. As usedherein, a “soluble P-selectin polypeptide” includes a P-selectinpolypeptide comprising amino acid sequences corresponding to theextracellular domain of a P-selectin protein, or a fragment thereof. Thenucleic acid and amino acid sequences of P-selectin proteins have beendescribed (see, for example, Sanders, W. E. et al. (1992) Blood80:795-800; and GenBank Accession Numbers NM_(—)003005 and M25322(human); GenBank Accession Numbers NM_(—)013114 and L23088 (rat);GenBank Accession Numbers NM_(—)011347 and M87861 (mouse); and GenBankAccession Number L12041 (bovine)). In another embodiment, a solubleP-selectin polypeptide comprises at least a lectin domain, an EGF-likerepeat, and at least two complement-binding domains of a P-selectinprotein. In yet another embodiment, a soluble P-selectin polypeptidebinds to a P-selectin ligand, e.g., PSGL-1. In a preferred embodiment, asoluble P-selectin polypeptide of the invention is a soluble P-selectinfusion protein. In one embodiment, the P-selectin fusion protein is aP-selectin-Ig fusion protein comprising a signal sequence, a lectindomain, an EGF-like repeat, and at least two complement-binding domainsof a P-selectin protein operatively linked to the Fc region (hinge, C1and C2) of an immunoglobulin, e.g., human IgG1.

[0045] In a further embodiment of the invention, hemostasis is inducedin a subject by administering an isolated nucleic acid moleculecomprising a nucleotide sequence which encodes a soluble P-selectinpolypeptide. In yet another embodiment, a recombinant cell expressing asoluble P-selectin polypeptide is administered to a subject to inducehemostasis.

[0046] Another embodiment of the invention provides methods for inducinghemostasis in a subject presenting insufficient hemostatic function,such as a subject having, or at risk of developing a disorder associatedwith hypocoagulation. As used herein, the term “hypocoagulation” refersto a decreased ability or inability to form blood clots. Such disordersinclude hemorrhagic disorders, e.g., hemophilia (e.g., hemophilia A orB), and disorders resulting from a deficiency in clotting factors orplatelet ligands, e.g., a deficiency in von Willebrand's factorresulting in von Willebrand disease. The induction of a procoagulantstate would prevent or stop spontaneous bleeding and would also bebeneficial preceding surgical intervention in a patient, or to promotewound healing.

[0047] The methods of the present invention are also useful for thetreatment of a vasculature-associated disease. As used herein, a“vasculature-associated disease” is a disease having a pathology that isdependent on a vascular blood supply. Thus, it is contemplated thatachieving coagulation in the vasculature of the disease site, e.g., inthe intratumoral vasculature of a solid tumor, would prove beneficial.Such vasculature-associated diseases include benign and malignant tumorsor growths, such as BPH, diabetic retinopathy, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, neovascularglaucoma and psoriasis. Also included within this group are synovitis,dermatitis, endometriosis, angiofibroma, rheumatoid arthritis,atherosclerotic plaques, corneal graft neovascularization, hemophilicjoints, hypertrophic scars, osler-weber syndrome, pyogenic granulomaretrolental fibroplasia, scleroderma, trachoma, and vascular adhesions.

[0048] In one embodiment, an inducer of P-selectin activity, e.g.,soluble P-selectin, is administered in addition to therapies designed toinduce thrombosis of tumor blood vessels, in order to potentiate tumornecrosis. Such therapies utilize strategies for targeting coagulationfactors to the tumor vasculature, for example, as described in U.S. Pat.No. 5,877,289. Markers of tumor vasculature or stroma may bespecifically induced and then targeted using a binding ligand, such asan antibody. Exemplary inducible antigens include E-selectin,P-selectin, MHC Class II antigens, VCAM-1, ICAM-1, endoglin, ligandsreactive with LAM-1, vascular addressins and other adhesion molecules.

[0049] Moreover, the present invention provides a method for reducinghemostasis in a subject by administering an inhibitor of P-selectinactivity. The inhibition of hemostasis, e.g., clot formation, isdesirable in situations where vessel patency is of importance.

[0050] In an exemplary embodiment, an inhibitor of P-selectin activitydecreases the level of circulating soluble P-selectin in the subject.The inhibitor of P-selectin activity may act to decrease thetranslocation of P-selectin from a cellular storage pool to the cellsurface, or to decrease the proteolytic cleavage and release of solubleP-selectin from the surface of a cell expressing P-selectin, e.g., anendothelial cell or a platelet. In another embodiment, an inhibitor ofP-selectin activity decreases P-selectin gene expression by reducingeither gene transcription or translation. In a preferred embodiment, aninhibitor of P-selectin activity will preferentially reduce theexpression of an alternatively spliced isoform of the P-selectin geneencoding a soluble P-selectin polypeptide lacking the transmembranedomain. In yet another embodiment, an inhibitor of P-selectin activityacts as an antagonist, wherein it binds to a P-selectin ligand orreceptor (e.g., PSGL-1) and blocks the activity of a P-selectinpolypeptide on a P-selectin responsive cell. In one embodiment of theinvention, an inhibitor of P-selectin activity is an anti-P-selectinantibody. In another embodiment, an inhibitor of P-selectin activity isa soluble PSGL-1 polypeptide. PSGL-1 nucleic acids, polypeptides, andsoluble forms thereof are disclosed in U.S. Pat. No. 5,843,707.

[0051] Alternatively, the invention provides a method for reducinghemostasis in a subject by administering an isolated nucleic acidmolecule comprising a nucleotide sequence which is antisense, e.g.,complementary to, to a nucleotide sequence encoding a P-selectinpolypeptide.

[0052] Thus, the methods of the invention are useful for the treatmentor prevention of thrombotic disorders. As used herein, the term“thrombotic disorder” includes any disorder or condition characterizedby excessive or unwanted coagulation or hemostatic activity, or ahypercoagulable state. Thrombotic disorders include disorders diseasesinvolving platelet adhesion and thrombus formation, and may manifest asan increased propensity to form thromboses, e.g., an increased number ofthromboses, thrombosis at an early age, a familial tendency towardsthrombosis, and thrombosis at unusual sites. Examples of thromboticdisorders include, but are not limited to, thromboembolism, deep veinthrombosis, pulmonary embolism, stroke, myocardial infarction,miscarriage, thrombophilia associated with anti-thrombin III deficiency,protein C deficiency, protein S deficiency, resistance to activatedprotein C, dysfibrinogenemia, fibrinolytic disorders, homocystinuria,pregnancy, inflammatory disorders, myeloproliferative disorders,arteriosclerosis, angina, e.g., unstable angina, disseminatedintravascular coagulation, thrombotic thrombocytopenic purpura, cancermetastasis, sickle cell disease, and glomerular nephritis. In addition,inhibitors of soluble P-selectin expression or activity are administeredto prevent thrombotic events or to prevent re-occlusion during or aftertherapeutic clot lysis or procedures such as angioplasty or surgery.

[0053] Furthermore, measuring the level P-selectin activity in abiological sample, e.g., in blood, would provide diagnostic informationof a procoagulant state, e.g., the likelihood of a thrombotic orclotting event. Accordingly, in one embodiment, the invention provides amethod for diagnosing a procoagulant state in a subject by detecting anincreased level of circulating soluble P-selectin as compared with thelevels of soluble P-selectin in the blood of individual with clinicallyestablished normal levels of hemostatic activity. In another embodiment,the invention provides a method of identifying a subject having athrombotic disorder, or at risk for developing a thrombotic disorder, bydetecting the presence of increased levels of P-selectin activity (e.g.,increased levels of circulating soluble P-selectin).

[0054] As used herein, a “hemostatic disorder” includes a disorder orcondition characterized by aberrant or unwanted hemostatic activity. Ahemostatic disorder may result from excessive coagulant activity, e.g.,a thrombotic disorder, or it may result from insufficient coagulantactivity, e.g., a hemorrhagic disorder.

[0055] Furthermore, another aspect of the invention provides a methodfor identifying a compound capable of modulating hemostasis by assayingthe ability of the compound to modulate a P-selectin activity, e.g., theexpression of soluble P-selectin.

[0056] Various aspects of the invention are described in further detailin the following subsections.

[0057] I. Isolated P-selectin Proteins and Anti-P-selectin Antibodies

[0058] The methods of the invention include the use of isolatedP-selectin polypeptides, and biologically active portions thereof. Asused herein, a “P-selectin protein” or “P-selectin polypeptide” includesa soluble P-selectin polypeptide and a soluble P-selectin fusionprotein.

[0059] The genomic organization and coding sequence for human P-selectinhave been determined, and the cDNA has been cloned and sequenced (see,for example, GenBank Accession Numbers NM_(—)003005 and M25322). Inaddition, the sequences encoding rat (GenBank Accession NumbersNM_(—)013114 and L23088), mouse (GenBank Accession Numbers NM_(—)011347and M87861), and bovine (GenBank Accession Number L12041) P-selectinhave been disclosed. Furthermore, a comparison of the amino acidsequences and structural domains of human and mouse P-selectin isdisclosed in Sanders, WE et al. (1992) Blood 80:795-800.

[0060] Isolated soluble P-selectin proteins for use in the methods ofthe present invention preferably have an amino acid sequence that issufficiently identical to the amino acid sequence of a native P-selectinprotein. As used herein, the term “sufficiently identical” refers to anamino acid (or nucleotide) sequence which contains a sufficient orminimum number of identical or equivalent (e.g., an amino acid residuethat has a similar side chain) amino acid residues (or nucleotides) to aP-selectin amino acid (or nucleotide) sequence such that the polypeptideshares common structural domains or motifs, and/or a common functionalactivity with a native P-selectin protein. For example, amino acid ornucleotide sequences which share common structural domains have at least30%, 40%, or 50% identity, preferably 60% identity, more preferably70%-80%, and even more preferably 90-95% identity across the amino acidsequences of the domains and contain at least one, and more preferablytwo or more structural domains or motifs, are defined herein assufficiently identical. For example, a soluble P-selectin polypeptidemay comprise at least one or more of the following domains: a signalpeptide, a lectin domain, an EGF-like repeat, a complement bindingdomain, and a cytoplasmic domain. Furthermore, amino acid or nucleotidesequences which share at least 30%, 40%, or 50%, preferably 60%, morepreferably 70-80%, or 90-95% identity and share a common functionalactivity (e.g., a soluble P-selectin activity as described herein) aredefined herein as sufficiently identical. A P-selectin polypeptide maydiffer in amino acid sequence from the P-selectin polypeptides disclosedherein due to natural allelic variation or mutagenesis. Accordingly,isolated soluble P-selectin polypeptides having a P-selectin activitycan be used in the methods of the invention.

[0061] To determine the percent identity of two amino acid sequences orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-identical sequences can be disregarded for comparisonpurposes). In a preferred embodiment, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, even more preferably at least 60%,and even more preferably at least 70%, 80%, or 90% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0062] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4.

[0063] As used herein, a “biologically active portion” of a P-selectinpolypeptide (e.g., a soluble P-selectin polypeptide) includes a fragmentof a P-selectin polypeptide which retains a P-selectin polypeptideactivity. Typically, a biologically active portion of a P-selectinpolypeptide comprises at least one domain or motif with at least oneactivity of the P-selectin polypeptide, e.g., modulating hemostaticactivity. Biologically active portions of a P-selectin polypeptideinclude polypeptides comprising amino acid sequences sufficientlyidentical to or derived from the amino acid sequence of a P-selectinprotein, which include less amino acids than the full length P-selectinpolypeptide, and exhibit at least one activity of a soluble P-selectinpolypeptide. Biologically active portions of a P-selectin polypeptidecan be used as targets for developing agents which modulate a P-selectinpolypeptide activity, e.g., a hemostatic activity. A biologically activeportion of a P-selectin polypeptide comprises a polypeptide which can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a P-selectin polypeptide.

[0064] In one embodiment, P-selectin polypeptides can be isolated fromcells or tissue sources by an appropriate purification scheme usingstandard protein purification techniques. For example, a solubleP-selectin polypeptide can be isolated from the culture medium of cells,e.g., activated endothelial cells, that have been induced to shedP-selectin from the cell surface. In another embodiment, P-selectinpolypeptides are produced by recombinant DNA techniques. For example, asoluble P-selectin polypeptide can be isolated from a host celltransfected with a polynucleotide sequence encoding a soluble isoform ofP-selectin (e.g., an isoform of P-selectin lacking a transmembranedomain) or a soluble P-selectin fusion protein. Alternative torecombinant expression, a soluble P-selectin polypeptide can besynthesized chemically using standard peptide synthesis techniques.

[0065] An “isolated” or “purified” polypeptide or protein, orbiologically active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the P-selectin polypeptide is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.The language “substantially free of cellular material” includespreparations of P-selectin polypeptide in which the protein is separatedfrom cellular components of the cells from which it is isolated orrecombinantly produced. In one embodiment, the language “substantiallyfree of cellular material” includes preparations of P-selectin proteinhaving less than about 30% (by dry weight) of non-P-selectin protein(also referred to herein as a “contaminating protein”), more preferablyless than about 20% of non-P-selectin protein, still more preferablyless than about 10% of non-P-selectin protein, and most preferably lessthan about 5% non-P-selectin protein. When the P-selectin polypeptide orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

[0066] The language “substantially free of chemical precursors or otherchemicals” includes preparations of P-selectin polypeptide in which theprotein is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of P-selectin polypeptide having less than about30% (by dry weight) of chemical precursors or non-P-selectin chemicals,more preferably less than about 20% chemical precursors ornon-P-selectin chemicals, still more preferably less than about 10%chemical precursors or non-P-selectin chemicals, and most preferablyless than about 5% chemical precursors or non-P-selectin chemicals.

[0067] The methods of the invention may also use soluble P-selectinpolypeptides that are chimeric or fusion proteins. As used herein, asoluble P-selectin “chimeric protein” or “fusion protein” comprises asoluble P-selectin polypeptide operatively linked to a non-solubleP-selectin polypeptide. A “soluble P-selectin polypeptide” includes aP-selectin polypeptide that comprises amino acid sequences correspondingto the extracellular domain of a P-selectin protein, or a biologicallyactive portion thereof, whereas a “non-soluble P-selectin polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially homologous to a P-selectinpolypeptide, e.g., a protein which is different from the solubleP-selectin polypeptide and which is derived from the same or a differentorganism. Within a soluble P-selectin fusion protein the solubleP-selectin polypeptide may include, for example, all or a portion of theextracellular domain of a P-selectin protein. In a preferred embodiment,a soluble P-selectin fusion protein comprises at least a signalsequence, a lectin domain, an EGF-like repeat, and at least twocomplement-binding domains of a P-selectin protein. Within the fusionprotein, the term “operatively linked” is intended to indicate that thesoluble P-selectin polypeptide and the non-soluble P-selectinpolypeptide are fused in-frame to each other. The non-soluble P-selectinpolypeptide can be fused to the N-terminus or C-terminus of the solubleP-selectin polypeptide.

[0068] For example, in a preferred embodiment, the fusion protein is asoluble P-selectin-immunoglobulin fusion protein in which the Fc region,e.g., the hinge, C1 and C2 sequences, of an immunoglobulin, (e.g., humanIgG1) is fused to the C-terminus of the soluble P-selectin sequences.Selectin immunoglobulin chimeras can be constructed essentially asdescribed in WO 91/08298. Such fusion proteins can facilitate thepurification of recombinant soluble P-selectin polypeptides. In anotherembodiment, the fusion protein is a soluble P-selectin polypeptidecontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofsoluble P-selectin can be increased through use of a heterologous signalsequence.

[0069] The soluble P-selectin polypeptides and fusion proteins of theinvention can be incorporated into pharmaceutical compositions andadministered to a subject in vivo. In an exemplary embodiment, a solubleP-selectin polypeptide or fusion protein may be used to modulatehemostasis in a subject (e.g., induce a procoagulant state). In anotherembodiment, a soluble P-selectin polypeptide or fusion protein may beused to treat a hemostatic disorder, e.g., a hemorrhagic disorder. Inanother embodiment, a soluble P-selectin polypeptide or fusion proteinmay be used to treat a vasculature-associated disease. Use of solubleP-selectin polypeptides and fusion proteins may also be usefultherapeutically for the treatment of disorders caused by, for example,(i) aberrant modification or mutation of a gene encoding a P-selectinprotein; (ii) mis-regulation of a P-selectin gene; and (iii) aberrantpost-translational modification of a P-selectin protein. In addition,the soluble P-selectin polypeptides and fusion proteins can be used toaffect the bioavailability of a P-selectin ligand, e.g., PSGL-1.

[0070] Moreover, the soluble P-selectin polypeptides and fusion proteinsof the invention can be used as immunogens to produce anti-P-selectinantibodies in a subject, to purify P-selectin ligands, and in screeningassays to identify molecules which modulate P-selectin activity, and/ormodulate the interaction of a P-selectin polypeptide with a P-selectinligand or receptor.

[0071] Preferably, a soluble P-selectin fusion protein of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A solubleP-selectin-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the solubleP-selectin polypeptide.

[0072] The methods of the present invention may also include the use ofvariants of a P-selectin polypeptide which function as either P-selectinagonists (mimetics) or as P-selectin antagonists. Variants of theP-selectin polypeptide can be generated by mutagenesis, e.g., discretepoint mutation or truncation of a P-selectin protein. An agonist of aP-selectin polypeptide can retain substantially the same, or a subset,of the biological activities of the naturally occurring form of aP-selectin polypeptide. An antagonist of a P-selectin polypeptide caninhibit one or more of the activities of a native form of the P-selectinpolypeptide by, for example, competitively modulating a P-selectinactivity (e.g., a hemostatic activity) of a P-selectin polypeptide.Thus, specific biological effects can be elicited by treatment with avariant of limited function. In one embodiment, treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of theP-selectin polypeptide.

[0073] In one embodiment, variants of a soluble P-selectin polypeptidewhich function as either soluble P-selectin agonists (mimetics) or assoluble P-selectin antagonists can be identified by screening mutants,e.g., truncation mutants, of a soluble P-selectin polypeptide forsoluble P-selectin polypeptide agonist or antagonist activity. Theactivity of a variant soluble P-selectin polypeptide, e.g., the abilityto modulate hemostatic activity, can be assessed in an animal model suchas the animal models described and exemplified herein, e.g., aP-selectin deficient mouse, or a von Willebrand factor deficient mouse.

[0074] An isolated P-selectin polypeptide, or a portion or fragmentthereof, can be used as an immunogen to generate antibodies that bindP-selectin using standard techniques for polyclonal and monoclonalantibody preparation (see, generally R. H. Kenneth, in MonoclonalAntibodies: A New Dimension In Biological Analyses, Plenum PublishingCorp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med.,54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36).Moreover, the ordinarily skilled artisan will appreciate that there aremany variations of such methods which also would be useful.

[0075] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-P-selectin antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with P-selectin to therebyisolate immunoglobulin library members that bind P-selectin. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. PCT International Publication No. WO92/18619; Dower et al. PCT International Publication No. WO 91/17271;Winter et al. PCT International Publication WO 92/20791; Markland et al.PCT International Publication No. WO 92/15679; Breitling et al. PCTInternational Publication WO 93/01288; McCafferty et al. PCTInternational Publication No. WO 92/01047; Garrard et al. PCTInternational Publication No. WO 92/09690; Ladner et al. PCTInternational Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res.19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

[0076] Additionally, recombinant anti-P-selectin antibodies, such aschimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, which can be made using standard recombinant DNAtechniques, can also be used in the methods of the present invention.Such chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example using methodsdescribed in Robinson et al. International Application No.PCTIUS86/02269; Akira, et al. European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.European Patent Application 173,494; Neuberger et al. PCT InternationalPublication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567;Cabilly et al. European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L.(1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214;Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525;Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J.Immunol. 141:4053-4060.

[0077] An anti-P-selectin antibody (e.g., a monoclonal antibody) can beused in the methods of the invention to modulate the expression and/oractivity of a soluble P-selectin polypeptide. Alternatively, an antibodyagainst a P-selectin ligand or receptor, e.g., PSGL-1, may be useful inthe methods of the invention. For example, an anti-PSGL-1 antibody maybe used to mimic the activity of soluble P-selectin. In one embodimentan activating anti-PSGL-1 antibody induces the release of microparticlescontaining tissue factor.

[0078] An anti-P-selectin antibody can also be used to isolate solubleP-selectin polypeptides or fusion proteins by standard techniques, suchas affinity chromatography or immunoprecipitation. An anti-P-selectinantibody can facilitate the purification of natural soluble P-selectinfrom cell cultures and of recombinantly produced soluble P-selectinexpressed in host cells. Moreover, an anti-P-selectin antibody can beused to detect soluble P-selectin polypeptide (e.g., in a blood sample)in order to evaluate the abundance and pattern of expression of thesoluble P-selectin polypeptide. Anti-P-selectin antibodies can be useddiagnostically to monitor protein levels in blood as part of a clinicaltesting procedure, e.g., to, for example, determine hemostatic activity,i.e., a procoagulant state. Detection can be facilitated by coupling(i.e., physically linking) the antibody to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, 131I, ³⁵S or ³H.

[0079] II. Isolated Nucleic Acid Molecules

[0080] The methods of the invention include the use of isolated nucleicacid molecules that encode P-selectin polypeptides (e.g., a solubleP-selectin polypeptide) or biologically active portions thereof. Thenucleotide sequences encoding human (GenBank Accession NumbersNM_(—)003005 and M25322), rat (GenBank Accession Numbers NM_(—)013114and L23088), mouse (GenBank Accession Numbers NM_(—)011347 and M87861),and bovine (GenBank Accession Number L12041) P-selectin have beendisclosed.

[0081] As used herein, the term “nucleic acid molecule” is intended toinclude DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

[0082] The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid moleculeencoding soluble P-selectin can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived. Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule, can be substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

[0083] A nucleic acid molecule of the present invention, e.g., a nucleicacid molecule encoding soluble P-selectin, a soluble P-selectin fusionprotein, or a portion thereof, can be isolated using standard molecularbiology techniques (e.g., as described in Sambrook, J., Fritsh, E. F.,and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

[0084] A nucleic acid of the invention can be amplified using cDNA, mRNAor alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to P-selectin nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

[0085] A nucleic acid fragment encoding a “biologically active portion”of a P-selectin polypeptide can be prepared by isolating a portion ofthe nucleotide sequence of a P-selectin gene having a P-selectinbiological activity (the biological activities, e.g., the hemostaticactivity, of soluble P-selectin are described herein), expressing theencoded portion of the P-selectin polypeptide (e.g., by recombinantexpression in vitro) and assessing the activity of the encoded portionof the P-selectin polypeptide.

[0086] The skilled artisan will further appreciate that changes can beintroduced by mutation into the nucleotide sequence encoding aP-selectin polypeptide, thereby leading to changes in the amino acidsequence of the encoded P-selectin polypeptide, without altering thefunctional ability of the P-selectin polypeptide. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of aP-selectin gene. A “non-essential” amino acid residue is a residue thatcan be altered from the wild-type sequence of a P-selectin polypeptidewithout altering the biological activity, whereas an “essential” aminoacid residue is required for biological activity. For example, aminoacid residues that are conserved among the P-selectin proteins fromdifferent species are predicted to be particularly unamenable toalteration.

[0087] Accordingly, the methods of the invention may include the use ofnucleic acid molecules encoding P-selectin polypeptides that containchanges in amino acid residues that are not essential for activity.

[0088] An isolated nucleic acid molecule encoding a P-selectinpolypeptide can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence of aP-selectin gene such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations can be introduced into a nucleic acid sequence by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a soluble P-selectinpolypeptide is preferably replaced with another amino acid residue fromthe same side chain family. Alternatively, in another embodiment,mutations can be introduced randomly along all or part of a P-selectincoding sequence, such as by saturation mutagenesis, and the resultantmutants can be expressed recombinantly and screened for biologicalactivity to identify mutants that retain activity, e.g., in an animalmodel described herein. In a preferred embodiment, a mutant solubleP-selectin polypeptide protein can be assayed for the ability tomodulate hemostatic activity.

[0089] In addition to the nucleic acid molecules encoding P-selectinpolypeptides described herein, another aspect of the invention pertainsto isolated nucleic acid molecules which are antisense thereto. An“antisense” nucleic acid comprises a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a protein, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can hydrogen bond to a sense nucleic acid. The antisense nucleicacid can be complementary to an entire P-selectin coding strand, or toonly a portion thereof. In one embodiment, an antisense nucleic acidmolecule is antisense to a “coding region” of the coding strand of anucleotide sequence encoding P-selectin. The term “coding region” refersto the region of the nucleotide sequence comprising codons which aretranslated into amino acid residues. In another embodiment, theantisense nucleic acid molecule is antisense to a “noncoding region” ofthe coding strand of a nucleotide sequence encoding P-selectin. The term“noncoding region” refers to 5′ and 3′ sequences which flank the codingregion that are not translated into amino acids.

[0090] Given the coding strand sequences encoding P-selectin, antisensenucleic acids of the invention can be designed according to the rules ofWatson and Crick base pairing. The antisense nucleic acid molecule canbe complementary to the entire coding region of P-selectin mRNA, butmore preferably is an oligonucleotide which is antisense to only aportion of the coding or noncoding region of P-selectin mRNA. Forexample, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of P-selectin mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest).

[0091] In yet another embodiment, the P-selectin nucleic acid moleculesof the present invention can be modified at the base moiety, sugarmoiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For example, thedeoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup B. et al. (1996)Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.,DNA mimics, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup B. et al.(1996) supra; Perry-OKeefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[0092] PNAs of P-selectin nucleic acid molecules can be used intherapeutic and diagnostic applications. For example, PNAs can be usedas antisense or antigene agents for sequence-specific modulation of geneexpression by, for example, inducing transcription or translation arrestor inhibiting replication. PNAs of P-selectin nucleic acid molecules canalso be used in the analysis of single base pair mutations in a gene,(e.g., by PNA-directed PCR clamping); as ‘artificial restrictionenzymes’ when used in combination with other enzymes, (e.g., SInucleases (Hyrup B. (1996) supra)); or as probes or primers for DNAsequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefesupra).

[0093] In another embodiment, PNAs of P-selectin can be modified, (e.g.,to enhance their stability or cellular uptake), by attaching lipophilicor other helper groups to PNAs, by the formation of PNA-DNA chimeras, orby the use of liposomes or other techniques of drug delivery known inthe art. For example, PNA-DNA chimeras of P-selectin nucleic acidmolecules can be generated which may combine the advantageous propertiesof PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g.,RNAse H and DNA polymerases), to interact with the DNA portion while thePNA portion would provide high binding affinity and specificity. PNA-DNAchimeras can be linked using linkers of appropriate lengths selected interms of base stacking, number of bonds between the nucleobases, andorientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA chimerascan be performed as described in Hyrup B. (1996) supra and Finn P. J. etal. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chaincan be synthesized on a solid support using standard phosphoramiditecoupling chemistry and modified nucleoside analogs, e.g.,5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can beused as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989)Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′DNA segment and a 3′PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

[0094] In other embodiments, the oligonucleotide may include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. WO88/09810) or the blood-brain barrier(see, e.g., PCT Publication No. WO89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) orintercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). Tothis end, the oligonucleotide may be conjugated to another molecule,(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent).

[0095] III. Recombinant Expression Vectors and Host Cells

[0096] The methods of the invention include the use of vectors,preferably expression vectors, containing a nucleic acid encoding aP-selectin polypeptide (or a portion thereof, e.g., a soluble P-selectinpolypeptide). As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the methods of the invention may includeother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

[0097] The recombinant expression vectors used in the methods of theinvention comprise a nucleic acid of the invention in a form suitablefor expression of the nucleic acid in a host cell, which means that therecombinant expression vectors include one or more regulatory sequences,selected on the basis of the host cells to be used for expression, whichis operatively linked to the nucleic acid sequence to be expressed.Within a recombinant expression vector, “operably linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner which allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel; Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cells and those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, and the like. The expression vectors used in the methods of theinvention can be introduced into host cells to thereby produce proteinsor peptides, including fusion proteins or peptides, encoded by nucleicacids as described herein (e.g., soluble P-selectin polypeptides, fusionproteins, and the like).

[0098] The recombinant expression vectors used in the methods of theinvention can be designed for expression of P-selectin polypeptides orfusion proteins in prokaryotic or eukaryotic cells, e.g., for use in themethods of the invention. For example, soluble P-selectin polypeptidesor fusion proteins can be expressed in bacterial cells such as E. coli,insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

[0099] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility and/or stability ofthe recombinant protein; and 3) to aid in the purification of therecombinant protein by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantprotein to enable separation of the recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase. Typical fusion expression vectors include pGEX (PharmaciaBiotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL(New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway,N.J.) which fuse glutathione S-transferase (GST), maltose E bindingprotein, or protein A, respectively, to the target recombinant protein.Purified P-selectin fusion proteins (e.g., soluble P-selectin-Ig) can beutilized to modulate hemostatic potential, as described and exemplifiedherein. In one embodiment, a soluble P-selectin fusion protein expressedin a retroviral expression vector of the present invention can beutilized to infect cells, e.g., hematopoietic cells, which aresubsequently transplanted into recipients. The hemostatic activity ofthe subject recipient is then examined after sufficient time has passed(e.g., six (6) weeks).

[0100] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

[0101] One strategy to maximize recombinant protein expression in E.coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Another strategy is to alterthe nucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al., (1992) NucleicAcids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

[0102] In another embodiment, the P-selectin expression vector is ayeast expression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

[0103] Alternatively, P-selectin polypeptides can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

[0104] In yet another embodiment, a nucleic acid used in the methods ofthe invention is expressed in mammalian cells using a mammalianexpression vector. Examples of mammalian expression vectors includepCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)EMBO J. 6:187-195). When used in mammalian cells, the expressionvector's control functions are often provided by viral regulatoryelements. For example, commonly used promoters are derived from polyoma,Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitableexpression systems for both prokaryotic and eukaryotic cells seechapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

[0105] In another embodiment, the recombinant mammalian expressionvector used in the methods of the invention is capable of directingexpression of the nucleic acid preferentially in a particular cell type(e.g., tissue-specific regulatory elements are used to express thenucleic acid). Tissue-specific regulatory elements are known in the art.Non-limiting examples of suitable tissue-specific promoters include thealbumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev.1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv.Immunol. 43:235-275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins(Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell33:741-748), neuron-specific promoters (e.g., the neurofilamentpromoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA86:5473-5477), endothelial cell-specific promoters (e.g., KDR/flkpromoter; U.S. Pat. No. 5,888,765), pancreas-specific promoters (Edlundet al. (1985) Science 230:912-916), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally-regulatedpromoters are also encompassed, for example the murine hox promoters(Kessel and Gruss (1990) Science 249:374-379) and the α-fetoproteinpromoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0106] The expression characteristics of an endogenous P-selectin genewithin a cell line or microorganism may be modified by inserting aheterologous DNA regulatory element into the genome of a stable cellline or cloned microorganism such that the inserted regulatory elementis operatively linked with the endogenous P-selectin gene. For example,an endogenous P-selectin gene which is normally “transcriptionallysilent”, i.e., a P-selectin gene which is normally not expressed, or isexpressed only at very low levels in a cell line or microorganism, maybe activated by inserting a regulatory element which is capable ofpromoting the expression of a normally expressed gene product in thatcell line or microorganism. Alternatively, a transcriptionally silent,endogenous P-selectin gene may be activated by insertion of apromiscuous regulatory element that works across cell types.

[0107] A heterologous regulatory element may be inserted into a stablecell line or cloned microorganism, such that it is operatively linkedwith an endogenous P-selectin gene, using techniques, such as targetedhomologous recombination, which are well known to those of skill in theart, and described, e.g., in Chappel, U.S. Pat. No. 5,272,071; PCTpublication No. WO 91/06667, published May 16, 1991.

[0108] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to P-selectin mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

[0109] Another aspect of the invention pertains to use of host cellsinto which a P-selectin nucleic acid molecule of the invention isintroduced, e.g., a P-selectin nucleic acid molecule within arecombinant expression vector or a P-selectin nucleic acid moleculecontaining sequences which allow it to homologously recombine into aspecific site of the host cell's genome. The terms “host cell” and“recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

[0110] A host cell can be any prokaryotic or eukaryotic cell. Forexample, a P-selectin polypeptide or fusion protein can be expressed inbacterial cells such as E. coli, insect cells, yeast or mammalian cells(such as hematopoietic cells, leukocytes, human umbilical veinendothelial cells (HUVEC), human microvascular endothelial cells(HMVEC), Chinese hamster ovary cells (CHO) or COS cells). Other suitablehost cells are known to those skilled in the art.

[0111] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

[0112] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a soluble P-selectin polypeptide or canbe introduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

[0113] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) aP-selectin polypeptide or fusion protein for use in the methods of theinvention. In one embodiment, a host cell (into which a recombinantexpression vector encoding a soluble P-selectin polypeptide or fusionprotein has been introduced) is cultured in a suitable medium such thata soluble P-selectin polypeptide or fusion protein is produced. Inanother embodiment, a soluble P-selectin polypeptide or fusion proteinis isolated from the medium or the host cell. A recombinant cellexpressing soluble P-selectin or a soluble P-selectin fusion protein canalso be administered to a subject to modulate hemostasis.

[0114] IV. Methods of Treatment

[0115] The present invention discloses methods for modulating hemostaticpotential by modulating P-selectin activity (e.g., the levels of solubleP-selectin). Accordingly, the present invention provides for bothprophylactic and therapeutic methods of treating a subject at risk of(or susceptible to) or having a hemostatic disorder, e.g., a disorderassociated with aberrant or unwanted hemostatic activity, or avasculature-associated disease. With regards to both prophylactic andtherapeutic methods of treatment, such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics. “Pharmacogenomics”, as used herein, refers to theapplication of genomics technologies such as gene sequencing,statistical genetics, and gene expression analysis to drugs in clinicaldevelopment and on the market. More specifically, the term refers thestudy of how a patient's genes determine his or her response to a drug(e.g., a patient's “drug response phenotype”, or “drug responsegenotype”.) Thus, another aspect of the invention provides methods fortailoring an individual's prophylactic or therapeutic treatment witheither soluble P-selectin or modulators of P-selectin activity accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

[0116] A. Prophylactic Methods

[0117] The assessment of P-selectin activity can used as a measure ofhemostatic activity. Accordingly, in one aspect, the invention providesa method for preventing in a subject, a hemostatic disorder, e.g., adisorder associated with an aberrant or unwanted hemostatic activity, ora vasculature-associated disease by administering to the subject amodulator of P-selectin activity, or a soluble P-selectin polypeptide.Subjects at risk for a hemostatic disorder or a vasculature-associateddisease can be identified by, for example, any or a combination ofdiagnostic or prognostic assays as described herein, e.g., by assessingP-selectin activity in a biological sample (i.e., plasma levels ofsoluble P-selectin). Administration of a prophylactic agent can occurprior to the manifestation of symptoms characteristic of the hemostaticdisorder, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type ofdisorder, for example, a soluble P-selectin polypeptide, or a modulatorof P-selectin activity, e.g., a P-selectin agonist or antagonist, can beused for treating the subject. The appropriate agent can be determinedbased on screening assays described herein.

[0118] B. Therapeutic Methods

[0119] Described herein are methods and compositions whereby hemostaticdisorders, vasculature-associated diseases, and symptoms thereof, may beameliorated. Certain hemostatic disorders, e.g., a hypocoagulable stateor a hemorrhagic disorder, are brought about, at least in part, by theabsence or reduction of hemostatic activity. As such, an increase inhemostatic activity would bring about the amelioration of diseasesymptoms. In addition, certain vasculature-associated diseases aresupported by a blood supply to the disease site, for example, to provideoxygen and nutrients. Similarly, the induction of a procoagulant statein the vasculature supplying such disease sites would provide abeneficial effect.

[0120] Alternatively, certain other hemostatic diseases, e.g., athrombotic disorder, are brought about, at least in part, by thepresence or increase in hemostatic activity. As such, an reduction inhemostatic activity would bring about the amelioration of diseasesymptoms.

[0121] Techniques for the modulating hemostasis using modulators ofP-selectin activity are discussed herein. Accordingly, another aspect ofthe invention pertains to methods of modulating hemostasis or hemostaticpotential for therapeutic purposes.

[0122] In an exemplary embodiment, the modulatory methods of theinvention involve administering a modulator of P-selectin activity, or asoluble P-selectin polypeptide. A modulator of P-selectin activityincludes an agent that modulates (e.g., induces or inhibits) one or moreactivities of P-selectin, or an agent that modulates soluble P-selectinexpression. A modulator of P-selectin activity can be an agent asdescribed herein, such as a nucleic acid or a protein, anaturally-occurring target molecule of a soluble P-selectin polypeptide(e.g., a P-selectin ligand), an anti-P-selectin antibody, a solubleP-selectin agonist or antagonist, a peptidomimetic of a solubleP-selectin agonist or antagonist, or other small molecule. In oneembodiment, the agent is an inducer of P-selectin activity. Examples ofsuch inducers include active soluble P-selectin polypeptides, a nucleicacid molecule encoding a soluble P-selectin polypeptide, and a solubleP-selectin mimetic, e.g., an activating anti-PSGL-1 antibody. In anotherembodiment, the agent is an inhibitor of P-selectin activity. Examplesof such inhibitors include antisense soluble P-selectin nucleic acidmolecules, anti-P-selectin antibodies, and soluble P-selectininhibitors, e.g., soluble PSGL-1. As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant or unwanted hemostatic activity. Inone embodiment, the method involves administering a modulator ofP-selectin activity. In another embodiment, the method involvesadministering a soluble P-selectin polypeptide or a nucleic acidencoding a soluble P-selectin polypeptide to induce hemostasis and/or aprocoagulant state.

[0123] (i) Methods for Inhibiting Soluble P-selectin Expression,Synthesis, or Activity

[0124] As discussed above, certain hemostatic disorders, e.g.,thrombotic disorders, may result from an increased or excessive level ofhemostatic activity. In such circumstances, hemostatic activity, e.g.,thrombosis, may have a causative or exacerbating effect on the diseasestate. In such cases, a reduction in hemostasis or hemostatic activitymay be achieved by reducing circulating levels of soluble P-selectin. Assuch, an inhibitor of P-selectin activity may be used in accordance withthe invention to reduce hemostasis. Such compounds may include, but arenot limited to, small organic molecules, peptides, antibodies, and thelike.

[0125] For example, compounds can be administered that compete withendogenous ligand for a soluble P-selectin polypeptide. The resultingreduction in the amount of ligand-bound soluble P-selectin polypeptidewill modulate hemostatic activity. Compounds that can be particularlyuseful for this purpose include, for example, soluble proteins orpeptides, such as peptides comprising one or more of the extracellulardomains, or portions and/or analogs thereof, of the P-selectin ligand,PSGL-1, including, for example, soluble fusion proteins such asIg-tailed fusion proteins. (For a discussion of the production ofIg-tailed fusion proteins, see, for example, U.S. Pat. No. 5,116,964).

[0126] In one embodiment, an inhibitor of P-selectin activity whichreduces or inhibits the translocation of P-selectin from cellularstorage pools to the cell surface, or which reduce or inhibit theproteolytic cleavage of cell surface P-selectin, can be effective inreducing circulating soluble P-selectin levels, and thus modulatinghemostatic activity. Alternatively, an inhibitor of P-selectin activitywhich reduces P-selectin gene expression (e.g., P-selectin genetranscription or translation), or the expression of an alternativelyspliced isoform of P-selectin lacking the transmembrane domain, can beused to reduce hemostasis.

[0127] Further, antisense and ribozyme molecules which inhibitexpression of the P-selectin gene may also be used in accordance withthe invention to inhibit hemostasis. Still further, triple helixmolecules may be utilized in inhibiting soluble P-selectin activity.

[0128] The antisense nucleic acid molecules of the invention aretypically administered to a subject or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding aP-selectin protein to thereby inhibit expression of the protein, e.g.,by inhibiting transcription and/or translation. The hybridization can beby conventional nucleotide complementarity to form a stable duplex, or,for example, in the case of an antisense nucleic acid molecule whichbinds to DNA duplexes, through specific interactions in the major grooveof the double helix. An example of a route of administration ofantisense nucleic acid molecules of the invention include directinjection at a tissue site. Alternatively, antisense nucleic acidmolecules can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, antisensemolecules can be modified such that they specifically bind to receptorsor antigens expressed on a selected cell surface, e.g., by linking theantisense nucleic acid molecules to peptides or antibodies which bind tocell surface receptors or antigens. The antisense nucleic acid moleculescan also be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

[0129] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

[0130] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes (described inHaselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave P-selectin mRNA transcripts to thereby inhibittranslation of P-selectin mRNA. A ribozyme having specificity for aP-selectin-encoding nucleic acid can be designed based upon thenucleotide sequence of a P-selectin cDNA. For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a P-selectin-encoding mRNA (see, for example, Cech etal. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).Alternatively, P-selectin mRNA can be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules(see, for example, Bartel, D. and Szostak, J. W. (1993) Science261:1411-1418).

[0131] P-selectin gene expression can also be inhibited by targetingnucleotide sequences complementary to the regulatory region of theP-selectin gene (e.g., the P-selectin promoter and/or enhancers) to formtriple helical structures that prevent transcription of the P-selectingene in target cells (see, for example, Helene, C. (1991) AnticancerDrug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15).

[0132] Antibodies that are both specific for the P-selectin protein andinterfere with its activity may also be used to modulate or inhibitP-selectin activity. Such antibodies may be generated, using standardtechniques, against the P-selectin protein itself or against peptidescorresponding to portions of the protein. Such antibodies include butare not limited to polyclonal, monoclonal, Fab fragments, single chainantibodies, or chimeric antibodies.

[0133] In instances where the target gene protein is intracellular,e.g., localized in storage granules, and whole antibodies are used,internalizing antibodies may be preferred. Lipofectin liposomes may beused to deliver the antibody or a fragment of the Fab region which bindsto the target epitope into cells. Where fragments of the antibody areused, the smallest inhibitory fragment which binds to the targetprotein's binding domain is preferred. For example, peptides having anamino acid sequence corresponding to the domain of the variable regionof the antibody that binds to the target gene protein may be used. Suchpeptides may be synthesized chemically or produced via recombinant DNAtechnology using methods well known in the art (described in, forexample, Creighton (1983), supra; and Sambrook et al. (1989) supra).Single chain neutralizing antibodies which bind to intracellular targetgene epitopes may also be administered. Such single chain antibodies maybe administered, for example, by expressing nucleotide sequencesencoding single-chain antibodies within the target cell population byutilizing, for example, techniques such as those described in Marasco etal. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[0134] In certain embodiments, antibodies that are specific for theextracellular domain of the P-selectin protein, for example, and thatinterfere with its activity, are particularly useful in modulatinghemostasis. Such antibodies are especially efficient because they canaccess the target domains directly from the bloodstream. Any of theadministration techniques described below which are appropriate forpeptide administration may be utilized to effectively administerinhibitory P-selectin antibodies to their site of action.

[0135] Antibodies for the modulation of P-selectin function aredisclosed in U.S. Pat. Nos. 6,033,667; 5,800,815; and 5,622,701.

[0136] The inhibitors of P-selectin, as described herein, may beadministered alone or in conjunction with other agents, compounds, orcompositions which are useful in reducing hemostasis or thrombosis,including, but not limited to, heparin, aspirin, and other anti-coagulants such as warfarin (Coumadin™), nicoumalone (Sintrom™), oranti-platelet aggregation agents such as inhibitors of αIIbβ3.

[0137] (ii) Methods for Restoring or Increasing P-selectin PolypeptideActivity

[0138] Certain hemostatic disorders, e.g., hemorrhagic disorders, mayresult from an reduced level of hemostatic activity. Moreover, theprogression of some vasculature-associated disorders is dependent on ablood supply to the disease site. In such circumstances, a reduction inor insufficient hemostatic activity, may have a causative orexacerbating effect on the disease state. In such cases, an increase inhemostasis or induction of a procoagulant state may be achieved by usingan inducer of P-selectin activity to increase P-selectin activity,preferably by increasing circulating levels of soluble P-selectin.

[0139] Described in this section are methods whereby the level ofsoluble P-selectin activity may be increased to levels wherein thesymptoms of hypocoagulation disorders or vasculature-associated diseasesare ameliorated. The level of soluble P-selectin polypeptide activitymay be increased, for example, by either increasing the level ofP-selectin gene expression, e.g., an alternatively spliced isoform ofP-selectin lacking the transmembrane domain, or by increasing the plasmalevel of active soluble P-selectin protein which is present.

[0140] For example, a soluble P-selectin polypeptide or fusion protein,at a level sufficient to ameliorate disease symptoms may be administeredto a patient exhibiting such symptoms. Any of the techniques discussedherein may be used for such administration. One of skill in the art willreadily know how to determine the concentration of effective, non-toxicdoses of the soluble P-selectin polypeptide, utilizing techniques suchas those described herein.

[0141] Additionally, RNA sequences encoding a soluble P-selectinpolypeptide may be directly administered to a patient exhibiting diseasesymptoms, at a concentration sufficient to produce a level of solubleP-selectin polypeptide such that disease symptoms are ameliorated. Anyof the techniques discussed below, which achieve intracellularadministration of compounds, such as, for example, liposomeadministration, may be used for the administration of such RNAmolecules. The RNA molecules may be produced, for example, byrecombinant techniques such as those described herein.

[0142] Further, subjects may be treated by gene replacement therapy. Oneor more copies of a gene encoding soluble P-selectin, or a solubleP-selectin fusion protein, that directs the production of a functionalsoluble P-selectin polypeptide or fusion protein, may be inserted intocells using vectors which include, but are not limited to adenovirus,adeno-associated virus, and retrovirus vectors, in addition to otherparticles that introduce DNA into cells, such as liposomes.Additionally, techniques such as those described above may be used forthe introduction of soluble P-selectin gene sequences into human cells.

[0143] Cells, preferably, autologous cells, containing solubleP-selectin expressing gene sequences may then be introduced orreintroduced into the subject at positions which allow for theamelioration of disease symptoms.

[0144] In one embodiment, inducers of P-selectin activity which increaseor enhance the translocation of P-selectin from cellular storage poolsto the cell surface, or which increase or enhance the proteolyticcleavage of cell surface P-selectin, can be effective in increasingcirculating soluble P-selectin levels, and thus modulating hemostaticactivity. Alternatively, compounds which stimulate P-selectin geneexpression (e.g., P-selectin gene transcription or translation), or theexpression of an alternatively spliced isoform of P-selectin lacking thetransmembrane domain, can be used to induce hemostasis. Furthermore,inducers of P-selectin activity which enhance P-selectin activity, e.g.,a soluble P-selectin agonist, may be used in accordance with theinvention to induce hemostasis. In another embodiment, inducers ofP-selectin activity which mimic P-selectin activity may be used tomodulate hemostatic activity. For example, an inducer of P-selectinactivity, e.g., an antibody, which binds to and activates a P-selectinligand or receptor on a cell can be used to modulate hemostasis. In oneembodiment, an antibody against PSGL-1, preferably an activatingantibody, binds to PSGL-1 on a cell and modulates hemostatic activity.In another embodiment, an inducer of P-selectin activity binds to aP-selectin ligand or receptor on a cell induces release ofmicroparticles containing tissue factor.

[0145] Such inducers of P-selectin activity may include, but are notlimited to, small organic molecules, peptides, antibodies, and the like.

[0146] Inducers of P-selectin activity, as described herein, may beadministered alone or in conjunction with other anti-hemorrhagic orpro-coagulant agents, compounds or compositions, including, but notlimited to Factor VIII, von Willebrand factor, platelets, the absorptionanalogue DDAVP, and fibrin, e.g., fibrin glue. In one embodiment,inducers of P-selectin activity as described herein may be administeredto a patient suffering from, for example, hemophilia A or von Willebranddisease where antibodies to Factor VIII have been developed by thepatient, thereby reducing the effectiveness of Factor VIII replacementtherapy alone.

[0147] C. Pharmacogenomics

[0148] A modulators of P-selectin activity, for example, as identifiedby a screening assay described herein, or a soluble P-selectinpolypeptide, can be administered to individuals to treat(prophylactically or therapeutically) hemostatic disorders associatedwith aberrant or unwanted hemostatic activity. In conjunction with suchtreatment, pharmacogenomics (i.e., the study of the relationship betweenan individual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a modulator of P-selectinactivity or a soluble P-selectin polypeptide, as well as tailoring thedosage and/or therapeutic regimen of treatment with a modulator ofP-selectin activity, or a soluble P-selectin polypeptide.

[0149] Pharmacogenomics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, for example, Eichelbaum, M. etal. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder,M. W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0150] One pharmacogenomics approach to identifying genes that predictdrug response, known as “a genome-wide association”, relies primarily ona high-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

[0151] Alternatively, a method termed the “candidate gene approach”, canbe utilized to identify genes that predict drug response. According tothis method, if a gene that encodes a drugs target is known (e.g.,P-selectin), all common variants of that gene can be fairly easilyidentified in the population and it can be determined if having oneversion of the gene versus another is associated with a particular drugresponse.

[0152] As an illustrative embodiment, the activity of drug metabolizingenzymes is a major determinant of both the intensity and duration ofdrug action. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

[0153] Alternatively, a method termed the “gene expression profiling”,can be utilized to identify genes that predict drug response. Forexample, the gene expression of an animal dosed with a drug (e.g., asoluble P-selectin polypeptide, or modulator thereof, of the presentinvention) can give an indication whether gene pathways related totoxicity have been turned on.

[0154] Information generated from more than one of the abovepharmacogenomics approaches can be used to determine appropriate dosageand treatment regimens for prophylactic or therapeutic treatment anindividual. This knowledge, when applied to dosing or drug selection,can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with asoluble P-selectin polypeptide or soluble P-selectin modulator.

[0155] VI. Screening Assays

[0156] The invention provides a method (also referred to herein as a“screening assay”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, small molecules(organic or inorganic) or other drugs) which modulate P-selectinactivity, and which may thus be used to modulate hemostatic potential.

[0157] These assays are designed to identify compounds, for example,that bind to a P-selectin polypeptide, e.g., a soluble P-selectinpolypeptide, bind to other proteins that interact with a P-selectinpolypeptide, and modulate the interaction of a P-selectin polypeptidewith other proteins, e.g., a P-selectin ligand, and thus modulateP-selectin activity. Screening assays can also be used to identifymodulators of P-selectin activity, for example, that regulate P-selectingene expression, the alternative splicing of the P-selectin geneencoding a soluble P-selectin isoform, the translocation of P-selectionfrom cellular storage pools to the cell surface, and the proteolyticcleavage of P-selection on the cell surface resulting in the release ofsoluble P-selectin. Moreover, screening assays can be used to identifyinducers of P-selectin activity, for example, that mimic the activity ofa P-selectin polypeptide, e.g., the binding of P-selectin to aP-selectin ligand or receptor, or the activity of P-selectin towards aP-selectin responsive cell. Such compounds may include, but are notlimited to, peptides, antibodies, or small organic or inorganiccompounds.

[0158] Compounds identified via assays such as those described hereinmay be useful, for example, for modulating hemostasis, and for treatinghemostatic disorders and/or vasculature-associated diseases. Ininstances whereby a hemostatic disorder or a vasculature-associateddisease results from an overall lower level of coagulation, usefulcompounds would bring about an effective increase in the level ofP-selectin activity, e.g., an inducer of P-selectin activity. In otherinstances wherein a hemostatic disorder results from an overallincreased level of coagulation or thrombosis, compounds that reduce thelevel of P-selectin activity would be beneficial, e.g., an inhibitor ofP-selectin activity. Cell and animal models for testing theeffectiveness of compounds identified by techniques such as thosedescribed in this section are discussed herein.

[0159] The test compounds can be obtained using any of the numerousapproaches in combinatorial library methods known in the art, including:biological libraries; spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des.12:145).

[0160] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

[0161] Libraries of compounds may be presented in solution (e.g.,Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner USP 5,223,409), spores (Ladner USP '409), plasmids (Cull et al.(1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith(1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici(1991) J. Mol. Biol. 222:301-310); (Ladner supra.).

[0162] In one embodiment, an assay is a cell-based assay comprisingcontacting a cell with a test compound and determining the ability ofthe test compound to modulate (e.g., induce or inhibit) P-selectinactivity. For example, a cell expressing a P-selectin ligand orreceptor, e.g., a leukocyte, is contacted with soluble P-selectinpolypeptide either alone or in the presence of a test compound, and theability of the test compound to modulate soluble P-selectin inducedrelease of microparticles containing tissue factor is determined, asdescribed herein. A similar cell-based assay could be used to identify acompound which mimics soluble P-selectin hemostatic activity, forexample, by assaying the test compound for the ability to induce therelease of microparticles containing tissue factor.

[0163] Furthermore, in another embodiment, a cell based assay can beused to determine the ability of the test compound to modulate thetranslocation of P-selectin to the cell surface, or to modulate theproteolytic cleavage of P-selectin from the cell surface. The presenceof P-selection on the surface of a cell can be assessed by standardtechniques, such as flow cytometry. The cleavage of P-selectin andconcurrent release of soluble P-selectin can be assessed by measuringthe level of membrane-associated P-selectin as compared to the level ofsoluble P-selectin in the culture medium.

[0164] In a further embodiment, modulators of P-selectin activity areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of soluble P-selectin mRNA or protein in thecell culture is determined by standard techniques. The level ofexpression of soluble P-selectin mRNA or protein in the presence of thecandidate compound is compared to the level of expression of solubleP-selectin mRNA or protein in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of solubleP-selectin activity based on this comparison. For example, whenexpression of soluble P-selectin mRNA or protein is greater(statistically significantly greater) in the presence of the candidatecompound than in its absence, the candidate compound is identified as ainducer of P-selectin activity. Alternatively, when expression ofsoluble P-selectin mRNA or protein is less (statistically significantlyless) in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of P-selectin activity.

[0165] In another embodiment, the ability of a test compound to modulatesoluble P-selectin binding to a receptor or ligand can also bedetermined, for example by coupling soluble P-selectin with aradioisotope or enzymatic label such that the binding of the solubleP-selectin can be determined by detecting labeled soluble P-selectin ina complex. For example, compounds (e.g., P-selectin polypeptides,P-selectin ligands) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, eitherdirectly or indirectly, and the radioisotope detected by direct countingof radioemmission or by scintillation counting. Compounds can further beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

[0166] Animal-based systems which act as models for hemostatic functionor disease, such as the animal models described and exemplified herein,e.g., P-selectin deficient mice and vWF deficient mice, include, but arenot limited to, non-recombinant and engineered transgenic animals.Models for studying vasculature-associated disease in vivo includeanimal models of tumori genesis, tumor metastasis, and arteriosclerosis.Models for studying thrombotic disorders in vivo include animal modelsof thrombosis such as those described in, at least, for example, Leadleyet al. (2000) J Phannacol Toxicol Methods 43: 101, and Dorffler-Melly,et al. (2000) Basic Res Cardiol 95:503.

[0167] The animal-based model systems may be used in a variety ofapplications, for example, as part of screening strategies designed toidentify compounds which are modulators of P-selectin activity. Thus,the animal-based models may be used to identify drugs, pharmaceuticals,therapies and interventions which may be effective in modulatinghemostasis and treating hemostatic disorders and vasculature-associateddiseases. For example, animal models may be exposed to a compound,suspected of exhibiting an ability to modulate P-selectin activity, andthe response of the animals to the exposure may be monitored byassessing hemostatic activity before and after treatment. Hemostaticactivity can be assessed using a clinically established test, e.g., atest of plasma clotting time, or using a method exemplified herein,e.g., fibrin formation in a perfusion chamber, plasma levels of solubleP-selectin and fibrinogen, hemorrhagic lesions in a local Schwartzmanreaction, tissue factor activity.

[0168] In another aspect, the invention pertains to a combination of twoor more of the assays described herein. For example, a modulator ofP-selectin activity can be identified using a cell-based assay, and theability of the agent to modulate P-selectin activity can be confirmed invivo, e.g., in an animal such as an animal model for hemostasis or ahemostatic disorder.

[0169] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further test a modulator of P-selectin activity asdescribed herein in an appropriate animal model for the ability tohemostatic potential. For example, an inducer or inhibitor of P-selectinactivity can be used in an animal model to determine the LD50 and theED50 in animal subjects, and such data can be used to determine the invivo efficacy, toxicity, or side effects of treatment with such apotential modulator of hemostatic activity.

[0170] With regard to intervention, any treatments which modulateP-selectin activity and/or hemostatic potential should be considered ascandidates for human therapeutic intervention. Dosages of test agentsmay be determined by deriving dose-response curves. Furthermore, thisinvention pertains to uses of newly identified modulators of P-selectinactivity for modulating hemostasis, as described herein.

[0171] Additionally, gene expression patterns may be utilized to assessthe ability of a compound, e.g., a modulator of P-selectin activity, tomodulate hemostasis. For example, the expression pattern of one or moregenes may form part of a “gene expression profile” or “transcriptionalprofile” which may be then be used in such an assessment. “Geneexpression profile” or “transcriptional profile”, as used herein,includes the pattern of mRNA expression obtained for a given tissue orcell type under a given set of conditions. Such conditions may include,but are not limited to, hemostatic disorders and/orvasculature-associated disease, including any of the control orexperimental conditions described herein, for example, in a localSchwartzman reaction, or in an animal model of P-selectin deficiency orvWF deficiency. Gene expression profiles may be generated, for example,by utilizing a differential display procedure, Northern analysis and/orRT-PCR. In one embodiment, P-selectin gene sequences may be used asprobes and/or PCR primers for the generation and corroboration of suchgene expression profiles.

[0172] Gene expression profiles may be characterized for known states,either hemostatic disease or normal, e.g., within the animal-based modelsystems described herein. Subsequently, these known gene expressionprofiles may be compared to ascertain the effect a test compound has tomodify such gene expression profiles, and to cause the profile to moreclosely resemble that of a more desirable profile.

[0173] For example, administration of a compound may cause the geneexpression profile of a hemostatic disorder model system to more closelyresemble the control system.

[0174] VI. Predictive Medicine

[0175] The present invention also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays, and monitoringclinical trials are used for prognostic (predictive) purposes to therebytreat an individual prophylactically. Accordingly, one aspect of thepresent invention relates to diagnostic assays for determiningP-selectin activity, e.g., soluble P-selectin expression in the contextof a biological sample (e.g., blood, serum, cells, tissue) to therebydetermine hemostatic activity, and to determine whether an individual isafflicted with a hemostatic disorder, or is at risk of developing ahemostatic disorder. The invention also provides for prognostic (orpredictive) assays for determining whether an individual is manifestinga procoagulant state. Such assays can be used for prognostic orpredictive purpose to modulate hemostasis, and thereby prophylacticallytreat an individual prior to the onset of a hemostatic disorder.

[0176] Another aspect of the invention pertains to monitoring theinfluence of agents (e.g., drugs, compounds) on hemostatic activity orprocoagulant state in clinical trials.

[0177] These and other agents are described in further detail in thefollowing sections.

[0178] A. Diagnostic Assays

[0179] The present invention encompasses methods for diagnostic andprognostic evaluation of hemostatic disease conditions, and for theidentification of subjects exhibiting a predisposition to suchconditions.

[0180] An exemplary method for detecting the presence or absencehemostatic activity in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample, e.g., a blood sample, with a compound or an agent capable ofdetecting P-selectin activity, e.g., a P-selectin binding substance thatdetects soluble P-selectin protein, such that the presence of P-selectinactivity is detected in the biological sample.

[0181] A preferred agent for detecting soluble P-selectin protein is anantibody capable of binding to soluble P-selectin protein, preferably anantibody with a detectable label. Antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,Fab or F(ab′)2) can be used. The term “labeled”, with regard to theprobe or antibody, is intended to encompass direct labeling of the probeor antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently labeled streptavidin.

[0182] The term “biological sample” is intended to include tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject. That is, the detection methodof the invention can be used to detect P-selectin activity in abiological sample in vitro as well as in vivo. In vitro techniques fordetection of P-selectin protein include enzyme linked immunosorbentassays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. For a detailed explanation of methods for carryingout Western blot analysis, see Sambrook et al, 1989, supra, at Chapter18. The protein detection and isolation methods employed herein may alsobe such as those described in Harlow and Lane, for example, (Harlow, E.and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.), which isincorporated herein by reference in its entirety.

[0183] Detection of P-selectin activity can be accomplished, forexample, by immunofluorescence techniques employing a fluorescentlylabeled antibody (see below) coupled with light microscopic, flowcytometric, or fluorimetric detection.

[0184] Often a solid phase support or carrier is used as a supportcapable of binding an antigen or an antibody. Well-known supports orcarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

[0185] One means for labeling an anti-P-selectin polypeptide specificantibody is via linkage to an enzyme and use in an enzyme immunoassay(EIA) (Voller, “The Enzyme Linked Immunosorbent Assay (ELISA)”,Diagnostic Horizons 2:1-7, 1978, Microbiological Associates QuarterlyPublication, Walkersville, Md.; Voller, et al., J. Clin. Pathol.31:507-520 (1978); Butler, Meth. Enzymol. 73:482-523 (1981); Maggio,(ed.) Enzyme Immunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa,et al., (eds.) Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzymewhich is bound to the antibody will react with an appropriate substrate,preferably a chromogenic substrate, in such a manner as to produce achemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby colorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

[0186] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling theantibodies or antibody fragments, it is possible to detect fingerprintgene wild type or mutant peptides through the use of a radioimmunoassay(RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays,Seventh Training Course on Radioligand Assay Techniques, The EndocrineSociety, March, 1986, which is incorporated by reference herein). Theradioactive isotope can be detected by such means as the use of a gammacounter or a scintillation counter or by autoradiography.

[0187] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0188] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

[0189] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

[0190] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0191] Furthermore, in vivo techniques for detection of P-selectinprotein include introducing into a subject a labeled anti-P-selectinantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

[0192] In one embodiment, the biological sample contains proteinmolecules from the test subject. A preferred biological sample is ablood sample isolated by conventional means from a subject (e.g.,venipuncture).

[0193] Moreover, it will be understood that any of the above methods fordetecting soluble P-selectin can be used to monitor the course oftreatment or therapy.

[0194] In another embodiment, the methods further involve obtaining acontrol biological sample from a control subject, contacting the controlsample with a compound or agent capable of P-selectin activity, e.g.,soluble P-selectin, such that the presence of P-selectin activity isdetected in the biological sample, and comparing the presence ofP-selectin activity in the control sample with the presence ofP-selectin activity in the test sample, to thereby assess hemostaticactivity.

[0195] In one embodiment, an increased level of P-selectin activity isindicative of increased hemostatic activity, e.g., a procoagulant state.In another embodiment, a decreased level of P-selectin activity isindicative of decreased hemostatic activity, e.g., a hypocoagulablestate.

[0196] B. Prognostic Assays

[0197] The diagnostic methods described herein can furthermore beutilized to identify subjects having or at risk of developing ahemostatic disorder e.g., a disorder associated with aberrant orunwanted hemostatic activity (i.e., a thrombotic disorder, a hemorrhagicdisorder). As used herein, the term “aberrant” includes a level ofhemostatic activity which deviates from clinically established normallevels of hemostatic activity under defined physiological conditions.Aberrant hemostatic activity includes increased or decreased hemostaticactivity. As used herein, the term “unwanted” includes an unwantedphenomenon involved in a biological response such as hemorrhage orthrombosis. For example, the term unwanted includes hemostatic activitywhich is undesirable in a subject.

[0198] The assays described herein, such as the preceding diagnosticassays or the following assays, can be utilized to identify a subjecthaving or at risk of developing a hemostatic disorder. Thus, the presentinvention provides a method for identifying a hemostatic disorderassociated with aberrant or unwanted hemostatic activity in which a testsample is obtained from a subject and P-selectin activity is detected,wherein the presence of aberrant or unwanted P-selectin activity isdiagnostic for a subject having or at risk of developing a hemostaticdisorder. As used herein, a “test sample” refers to a biological sampleobtained from a subject of interest. For example, a test sample can be abiological fluid (e.g., serum), cell sample, or tissue.

[0199] Furthermore, the prognostic assays described herein can be usedto determine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a hemostatic disorder.For example, such methods can be used to determine whether a subject canbe effectively treated with an agent for a hemorrhagic disorder or athrombotic disorder. Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a hemostatic disorder, e.g., a disorder associated with aberrant orunwanted hemostatic activity, in which a test sample is obtained andP-selectin activity is detected (e.g., wherein the level of P-selectinactivity is diagnostic for a subject that can be administered the agentto treat a hemostatic disorder).

[0200] Furthermore, any cell type or tissue in which P-selectin activityis expressed may be utilized in the prognostic assays described herein.

[0201] C. Monitoring of Effects During Clinical Trials

[0202] The present invention provides methods for evaluating theefficacy of drugs and monitoring the progress of patients involved inclinical trials for the treatment of hemostatic disorders.

[0203] Monitoring the influence of agents (e.g., drugs) on P-selectinactivity can be applied not only in basic drug screening, but also inclinical trials. For example, the effectiveness of an agent determinedby a screening assay as described herein to induce P-selectin activitycan be monitored in clinical trials of subjects exhibiting decreased orinsufficient hemostatic activity. Alternatively, the effectiveness of anagent determined by a screening assay to inhibit P-selectin activity canbe monitored in clinical trials of subjects exhibiting increasedhemostatic activity, e.g., thrombosis or a procoagulant state. In suchclinical trials, P-selectin activity can be used as a “read out” ormarker of hemostatic activity. In addition, the level of P-selectinactivity may be used as a read out of a particular drug or agent'seffect on a hemostatic activity.

[0204] In a preferred embodiment, the present invention provides amethod for monitoring the effectiveness of treatment of a subject withan modulator of P-selectin activity (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate identified by the screening assays described herein)including the steps of (i) obtaining a pre-administration sample from asubject prior to administration of the agent; (ii) detecting the levelof P-selectin activity in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of P-selectin in the post-administration samples; (v)comparing the level of P-selectin activity in the pre-administrationsample with that in the post administration sample or samples; and (vi)altering the administration of the agent to the subject accordingly. Forexample, increased administration of an inducer of P-selectin activitymay be desirable to increase P-selectin activity to higher levels thandetected, i.e., to increase the effectiveness of the agent to promotehemostasis. Alternatively, increased administration an inhibitor ofP-selectin activity may be desirable to lower P-selectin activity tolower levels than detected, i.e. to increase the effectiveness of theagent to downregulate hemostasis. According to such an embodiment,P-selectin activity may be used as an indicator of the effectiveness ofan agent, even in the absence of an observable phenotypic response.

[0205] VII. Pharmaceutical Compositions

[0206] Active compounds for use in the methods of the invention can beincorporated into pharmaceutical compositions suitable foradministration. As used herein, the language “active compounds” includesnucleic acid molecules encoding soluble P-selectin, soluble P-selectinproteins, and active fragments thereof, and anti-P-selectin antibodies.Active compounds also include modulators of soluble P-selectin activity,e.g., inducers and inhibitors, identified compounds that modulateP-selectin gene expression, synthesis, and/or activity, or compoundsthat mimic P-selectin activity, e.g., an anti-PSGL-1 antibody. Suchcompositions typically comprise the compound, nucleic acid molecule,protein, or antibody and a pharmaceutically acceptable carrier. As usedherein the language “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

[0207] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, ophthalmic, and rectal administration, including directinstallation into a disease site. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0208] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0209] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a inducer or inhibitor of P-selectin activity, asoluble P-selectin fusion protein) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

[0210] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0211] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0212] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0213] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0214] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0215] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals. In one embodiment, a “therapeuticallyeffective dose” refers to that amount of an active compound sufficientto result in modulation of hemostasis or hemostatic potential. Inanother embodiment, a therapeutically effective dose refers to an amountof an active compound sufficient to result in amelioration of symptomsof a hemostatic disorder or a vasculature-associated disease. In yetanother embodiment, a therapeutically effective dose refers to thatamount of an active compound sufficient to modulate the level and/oractivity of soluble P-selectin.

[0216] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0217] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0218] As defined herein, a therapeutically effective amount of proteinor polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

[0219] In a preferred example, a subject is treated with antibody,protein, or polypeptide in the range of between about 0.1 to 20 mg/kgbody weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody, protein, orpolypeptide used for treatment may increase or decrease over the courseof a particular treatment. Changes in dosage may result and becomeapparent from the results of diagnostic assays as described herein.

[0220] The present invention encompasses active agents which modulatesoluble P-selectin expression or activity. An agent may, for example, bea small molecule. For example, such small molecules include, but are notlimited to, peptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds. It is understood that appropriatedoses of small molecule agents depends upon a number of factors withinthe ken of the ordinarily skilled physician, veterinarian, orresearcher. The dose(s) of the small molecule will vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which thecomposition is to be administered, if applicable, and the effect whichthe practitioner desires the small molecule to have upon the nucleicacid or polypeptide of the invention.

[0221] Exemplary doses include milligram or microgram amounts of thesmall molecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

[0222] In certain embodiments of the invention, a modulator ofP-selectin activity is administered in combination with other agents(e.g., a small molecule), or in conjunction with another, complementarytreatment regime. For example, in one embodiment, an inducer ofP-selectin activity is used to treat a vasculature-associated disease.In the instance where the vasculature-associated disease is a tumor, thesubject may be treated with an inducer of P-selectin activity, andfurther treated with a molecule effective to induce a procoagulant statein tumor associated vasculature, e.g., a molecule comprising a firstbinding region that binds to a component of a tumor cell or tumorassociated vasculature (e.g., VCAM-1) operatively linked to acoagulation factor or a second binding region that binds to acoagulation factor, thereby increasing effectiveness of the treatment atthe disease site. The vessels at the disease site in othervasculature-associated diseases may be similarly targeted with acoagulation factor or pro-coagulant agent, such that the specificity andeffectiveness of the inducer of P-selectin activity is enhanced. Inanother embodiment, an inhibitor of P-selectin activity may be used inconjunction with anti-coagulant agents (e.g., integrin inhibitors,aspirin, heparin) in the treatment of thrombotic disorders, such asrestenosis following medical intervention.

[0223] Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. The conjugates of the invention can be used formodifying a given biological response, and the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, acoagulation factor such as tissue factor; a protein such as vascularendothelial growth factor (“VEGF”), platelet derived growth factor, andtissue plasminogen activator; biological response modifiers such as, forexample, lymphokines, cytokines and growth factors; or a toxin.

[0224] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2^(nd) Ed.), Robinson et al. (eds.), pp.623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

[0225] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0226] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0227] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures, are incorporated herein byreference.

EXAMPLES Example 1 Hemostatic Potential in Animals with Increased Levelsof Soluble P-selectin

[0228] Transgenic mice that express P-selectin lacking the cytoplasmicdomain (ΔCT mice) have been generated by gene replacement throughhomologous recombination in embryonic stem cells (Hartwell, D. W. et al.J Cell Biol (1998) 143:1129-1141). These mutant animals display anelevated level of soluble P-selectin in the plasma.

[0229] This example describes studies of the hemostatic potential in ΔCTmice as compared to wild type controls.

[0230] A. Fibrin Formation in a Perfusion Chamber

[0231] Fibrin formation of non-anticoagulated blood from wild type (WT),ΔCT mice, and P-selectin deficient (P-sel −/−) mice (Mayadas, T. N. etal. Cell (1993) 74:541-554) was compared ex vivo in a perfusion chamber.Leukocyte rolling and neutrophil extravasation, as well as hemostasisare compromised in these mice (Subramaniam, M. et al. Blood (1996)87:1238-1242).

[0232] Briefly, glass capillary tubes (0.56 mm inner diameter) werecoated with 1 mg/ml human fibrillar type III collagen (Sigma, St. Louis)as previously described (Andre, P. et al. Arterioscler Thromb Vasc Biol(1996) 16:56-63). Mice were anesthetized with 2.5% tribromoethanol (0.15ml/10 g). Non-anticoagulated blood was collected directly from the venacava of the mice using a butterfly 25 G, and perfused through thecollagen coated perfusion chamber using silastic tubing. A flow rate of220 μl/minute was established for 2 minutes by a syringe pump (HarvardApparatus) mounted distal to the chamber, resulting in a shear rate of212 s⁻¹, according to the equation: γ=4Q/πr³. Immediately after theblood perfusion, the thrombotic deposits formed onto the collagensurface were rinsed for 20 seconds with PBS and fixed in an ice cold2.5% cacodylate buffered glutaraldehyde (pH 7.4) at the same shear rate.The perfusion chamber was then removed from the flow system and fixed ina freshly prepared fixative buffer for 24 hours at 4° C. En facevisualization of the thrombotic deposits was performed under lightmicroscopy after epon embedding.

[0233]FIG. 1 is a photograph of en face examination of the thromboticdeposits formed after a 2 minute non-anticoagulated blood perfusion(blood flow, left to right). The white arrow indicates platelet richthrombus; the black arrow indicates fibrin tail formed distally theplatelet thrombus. As shown in FIG. 2, in 4 out of 11 perfusion chambersperformed with wild type animals (one perfusion chamber per animal), afibrin tail was found distally to the platelet aggregate. In 8 out of 9perfusion chambers performed in ΔCT mice, a fibrin tail was present. Inaddition, the fibrin tail from the ΔCT mice was significantly longerthan that observed in the wild type mice. None of the perfusion chambersperformed with P-selectin deficient blood exhibited a fibrin tail. Thestatistical comparison between fibrin formation in the 3 genotypes wasperformed using the Log rank test. A Student's t test was used tocompare the length of the fibrin tail.

[0234] B. Levels of Soluble P-selectin and Fibrinogen in Plasma

[0235] The level of soluble P-selectin in plasma was measured using amodified sandwich ELISA procedure as previously described (Hartwell, D.W. et al. J Cell Biol (1998) 143:1129-1141). Briefly, plasma samples ofwild type (WT) and ΔCT mice were incubated for 2 hours at 37° C. withmonoclonal anti-mouse P-selectin antibody (RB 40.34, Pharmingen Corp.,San Diego, Calif.)-coated plates. After washing, a biotinylated rabbitanti-P-selectin antibody (Pharmingen Corp., San Diego, Calif.) was addedto the wells and incubated for 2 hours. ExtrAvidin-conjugated alkalinephosphatase was added and the activity was revealed with p-nitrophenylphosphate (Sigma Chemical Co., St Louis, Mo.). Plates were read at 405nm in an Epson LX-300 ELISA reader (Dynatech Laboratories, Chantilly,Va.). The plasma level of fibrinogen was measured according to the SigmaDiagnostics Procedure No. 886 (St. Louis, Mo.) and expressed in mg/dL.

[0236] As shown in Table 1 below, a 3-fold increase in the level ofsoluble P-selectin was found in the plasma of ΔCT mice compared withwild type mice. In contrast, no significant difference was observed inthe plasma fibrinogen levels in these animals. TABLE 1 SolubleP-selectin in plasma Fibrinogen level in plasma (μg/ml) n (mg/dl) N WT0.34 4 WT 367 ± 24 13 ΔCT 1.05 4 ΔCT 344 ± 14 13

[0237] C. Hemorrhagic Lesions in a Local Shwartzman Reaction

[0238] Local Shwartzman reaction is a hemorrhagic and necrotic lesioninduced by endotoxin and cytokines, and is a prototypic model for theinterrelation between the inflammatory and hemostatic systems. Briefly,12 to 14 week old age-matched male wild type (WT) and ΔCT mice wereprimed on day 0 by a subcutaneous injection of Escherichia coli LPS055:B5 (Difco Laboratories, Detroit, Mich.) at 100 μg/mouse in 0.1 ml ofsterile phosphate buffered saline (PBS). Twenty four hours later (day1), recombinant TNF-A (Genzyme, Cambridge, Mass.) at 0.3 μg/mouse wasinjected at the same skin site, as described (Subramaniam, M et al.Blood (1996) 87:1238-1242). On day 2, the hemorrhagic lesions wereexamined and scored on a scale of 0 to 4 without knowledge of the mousegenotypes. Hematoxylin-eosin stained paraffin sections were preparedfrom the lesion site and the degree of inflammatory cell infiltration aswell as hemorrhage were scored microscopically, on a scale of 0 to 4.

[0239] Macroscopic and microscopic evaluation of the injection sitesrevealed that after 48 hours, the average size of the hemorrhagiclesions in ΔCT mice was about 50% of that in the wild type (see FIG. 3).A highly significant reduction of the hemorrhage was also observed inwild type animals perfused with soluble P-selectin-Ig (1 μg/g;Pharmingen Corp., San Diego, CA) injected 1 hour prior to TNFa challengeas compared to those injected with human IgG1 (Sigma Chemical Co., StLouis, Mo.).

[0240] D. Fibrin Deposition in a Local Shwartzman Reaction

[0241] Paraffin sections from the Shwartzman lesion site of wild typemice injected with human IgG1 or soluble P-selectin, as described above,were de-paraffinized, sequentially blocked with avidin D solution andbiotin blocking solution (Vector, Burlingame, Calif.), and then stainedwith a rabbit anti-human fibrinogen antibody (1:1000 dilution; A0080,Dako, Carpinteria, Calif.) which cross-reacts with mousefibrin/fibrinogen. Sections were then sequentially treated with abiotinylated goat anti-rabbit secondary antibody (Zymed LaboratoriesInc., South San Francisco, Calif.), and an ABC mix solution (Vector,Burlingame, Calif.). Development was done by treating the sections withan AEC substrate kit for horseradish peroxidase (Vector, Burlingame,Calif.). Sections were counterstained with hematoxylin for 5 minutes.

[0242] All vessels which presented fibrin staining outside of the vesselwall were classified as “leakage”. Vessels which presented fibrinstaining on the luminal surface of the endothelial cells without fibrinoutside the vessel wall were classified as “ring”. The results are shownin FIG. 4. Wild type animals injected with soluble P-selectin exhibiteda significant decrease in the percentage of “leakage” vessels, and anincrease in the percentage of “ring” vessels, as compared with animalsperfused with human IgG1.

[0243] E. Plasma Clotting Time

[0244] The plasma clotting time of wild type mice, either untreated, orinfused with either human IgG1 (control) or soluble P-selectin(s-P-sel), P-selectin deficient, and ΔCT mice, either untreated orinfused with human recombinant PSGL-1 (r-PSGL-1), was determined asfollows. Briefly, 1 ml of blood was drawn from the retro-orbital venousplexus using plain microhematocrit capillary tubes and collected intopolypropylene tubes containing 10% final volume of acid-citrate-dextrose(ACD: 38 mM citric acid, 75 mM trisodium citrate, 100 mM dextrose).Platelet poor plasma was prepared by centrifugation at 1,500 g for 25minutes, followed by centrifugation at 15,000 g for 2 minutes to removeany contaminating cells from the plasma. Plasma clotting time wasinduced under stirring conditions (800 rpm) at 37° C. in an aggregometerby adding an equal volume of pre-warmed 20 mM CaCl₂ solution to theplasma in a siliconized tube.

[0245] As shown in FIG. 5, ΔCT mice presented a significant reduction ofthe clotting time compared with wild type mice. In addition, asignificant increase of the clotting time was observed on day 4 in ΔCTmice injected intravenously (on days 0 and 2) with human recombinantPSGL-1 IgG (10 mg/kg). In contrast, injection of soluble P-selectin inwild type mice significantly reduced the clotting time compared with theIgG treated control group.

[0246] F. Microparticles in Mouse Plasma

[0247] The levels of microparticles circulating in vivo in wild typemice, untreated, or infused with either human IgG1 (control) or solubleP-selectin (s-P-sel), and in ΔCT mice was determined as follows.Briefly, platelet poor plasma was prepared as described above.Subsequently, 300 μl of platelet poor plasma was collected per mouse,and three samples of platelet poor plasma from mice of the same genotypewere pooled together, diluted 1:3 with buffer (10 mmol/L HEPES, 5 mmol/LKCl, 1 mmol/L MgCl₂, 136 mmol/L NaCl, pH 7.4), and centrifuged for 1.5hours at 100,000 g. The supernatant was discarded and the pellet ofmicroparticles was resuspended in a fixed volume (120 μl) of the samebuffer.

[0248] Flow cytometric analysis was performed on a Becton-DickinsonFACSCalibur (Franklin Lakes, N.J.) with CellQuest software(Becton-Dickinson, San Jose, Calif.). The light scatters and fluorescentchannels were set at logarithmic gain (forward scatter was EOO with athreshold of 12 and sideward scatter was 300). To count the totalpopulation of microparticles, 30 μl aliquots were incubated for 15minutes in the dark with calcein AM (0.25 μg/ml; Molecular Probes,Eugene, Oreg.). The total number of events were counted for a setinterval of 10 seconds.

[0249]FIG. 6 shows that the number of microparticles was increased by1.9-fold in ΔCT mice compared with wild type animals. Furthermore, a2.7-fold increase in microparticles was obtained when wild type micewere injected intravenously with soluble P-selectin-Ig, as compared tohuman IgG1.

[0250] To identify the origin of the procoagulant activity,microparticle samples were stained for 20 minutes at room temperaturewith a sheep anti-rabbit tissue factor IgG (American Diagnostica Inc.,Greenwich, Conn.) which recognizes mouse tissue factor (5 μg/ml finalconcentration). A FITC-conjugated rabbit anti-sheep IgG (1:1000dilution; Zymed Laboratories Inc., South San Francisco, Calif.) was usedas a secondary antibody. As controls, an identical concentration ofcontrol IgG antibodies were used (rat IgG, Sigma Chemical Co., St.Louis, Mo.; FITC-conjugated sheep IgG, Caltag Laboratories, Burlingame,Calif.). The microparticles were analyzed by flow cytometry.

[0251]FIG. 7 shows that there are an increased number of microparticlesexpressing tissue factor in the plasma of ΔCT mice.

[0252] G. Treatment of ΔCT Mice with Soluble PSGL-Ig

[0253] Soluble PSGL-Ig infusion decreases the pro-coagulant phenotype ofΔCT mice as shown by a significant decrease in the number ofmicroparticles and a prolonged clotting time of plasma. Infusion ofcontrol Ig had no such effect.

[0254] Plasma clotting time was determined as described above. Foranalysis of microparticles in plasma of ΔCT mice treated with PSGL-Ig,200 μl of blood was collected by retro-orbital puncture on day 0.Platelet-poor plasma was obtained, and 40 l was diluted in 260 μl PBSand immediately analyzed for microparticle number by FACS. Mice werethen infused i.v. (days 0 and 2) with 10 mg/kg PSGL-Ig or control Ig. Onday 4, 200 μl of blood was collected from the other eye, and the numberof microparticles was determined.

[0255]FIG. 11A shows the number of microparticles present in 40 μl ofΔCT plasma, before (open bars) and after (filled bars) two infusions ofPSGL-Ig and control Ig in ΔCT mice (*=p<0.05).

[0256]FIG. 11B shows that the clotting time at the end of the experiment(e.g., after 4 days) was significantly longer in mice treated withsoluble PSGL-Ig (filled bar) than in control Ig treated group (open bar)(*=p<0.05). These data show that inhibition of soluble P-selectindecreases the pro-coagulant state in vivo.

Example 2 Activity of Soluble P-selectin in von Willebrand FactorDeficient Mice and Mice with Hemophilia A

[0257] von Willebrand factor (vWF) deficient mice have only about 20% ofthe wild type level of factor VIII (anti-hemophilia factor), and thushave difficulty making fibrin clots (Denis, C. et al. Proc Natl Acad SciUSA (1998) 95:9524-9529). Mice with hemophilia A are lacking factor VIIIcompletely (Bi, L. et al. (1995) Nature Genetics 10:119-121. Thisexample describes the hemostatic activity of soluble P-selectin in theseanimals.

[0258] A. Tissue Factor Activity in Platelet Poor Plasma

[0259] Platelet poor plasma was prepared from pooled plasma of vWFdeficient mice (vWF −/−) infused with soluble P-selectin-Ig (n=2) orIgG1 (control; n=3). Microparticles were prepared by repeatedcentrifugation of platelet poor plasma. Briefly, the firstcentrifugation step at 12,000 g for 2 minutes was performed to removeany contaminating cells. The supernatant was then diluted in a 20 mMHEPES, 1 mM EDTA, pH7.2 solution and ultracentrifuged at 200,000 g for90 minutes. The supernatant was discarded, and the pelletedmicroparticles were resuspended (½ of the initial volume) in a 10 mMHEPES, 136 mM NaCl, pH7.4 solution. Determination of tissue factoractivity of the microparticle solution was measured through its abilityto promote the activation of factor X (150 nM) by factor VIla (5 nM) inthe presence of 1 mM CaCl₂. The reaction was allowed to proceed for 20minutes at 37° C. and was stopped by the addition of an excess of EDTA(5 mM final concentration). A chromogenic substrate of factor Xa,Spectrozyme® fXa, was added at a final concentration of 0.3 mM. Thechange in absorbance at 405 nm versus time was immediately recordedusing a plate reader equipped with kinetics software (DYNEXTechnologies, Inc.). The linear changes in absorbance directly correlatewith the concentration of factor Xa generated in the assay.

[0260] As shown in Table 2 below, the tissue factor activity of thesolution of microparticles from vWF deficient mice infused with solubleP-selectin-Ig was 2.1 fold higher than that of control mice infused withIgG1. TABLE 2 Tissue Factor (Xa) Activity in OD/minute vWF −/− + IgG1vWF −/− + soluble P-selectin-Ig 2.54 5.26

[0261] B. Procoagulant Microparticle Generation by Infusion of SolubleP-selectin-Ig

[0262] The levels of microparticles circulating in vivo in vWF deficientmice, infused with either human IgG1 (control) or soluble P-selectin-Ig(sP-sel-Ig) was determined as described above. FIG. 8 shows that thenumber of microparticles was increased when vWF deficient mice wereinjected intravenously with soluble P-selectin-Ig, as compared to humanIgG1 (control).

[0263] C. Prothrombin Clotting Time

[0264] Prothrombin clotting time (PT) is a global coagulation screeningtest. It involves extrinsic pathway of coagulation starting withactivation of TF-VII(a) complex. PT time is measured in prewarmed (37°C.) platelet poor plasma after adding thromboplastin as a source oftissue factor, and Ca²⁺.

[0265] Diluted prothrombin time was measured when pooled platelet poorplasma sample (0.1 ml) was mixed with 0.2 ml of diluted rabbit brainthromboplastin (IL TEST PT). Clotting time was determined usingphotometry detection of the first fibrin threads formed. FIG. 9 shows aprolonged prothrombin clotting time in vWF deficient plasma (vWF 4-)compared with wild type (wt) when the thromboplastin concentrationdecreased. This can be explained by the 20% of normal level of factorVIII found in the vWF deficient mice.

[0266] Clotting time of vWF deficient mice infused with either solubleP-selectin-Ig or IgG1 (control) was tested at the high dilution ofthromboplastin (1:20,000) because it is known that at that dilution,prothrombin clotting time is preferentially tissue factor dependent. Theinfusion of soluble P-selectin-Ig in vWF deficient mice shortened theprothrombin clotting time by 28% when compared with vWF deficient miceinfused with IgG1.

[0267] D. Bleeding Time

[0268] Bleeding time was measured as described by Dejana, et al. (1979)Thromb. Res. 15:199-201. Briefly, factor VIII-deficient mice wereinjected with 1.2 μg soluble P-selectin-Ig (P-sel-Ig) or human IgG1control per gram of mouse. Six hours later mice were put in arestrainer, and a distal 3-mm segment of the tail was severed with arazor blade. The tail was immediately immersed in 0.9% isotonic salineat 37° C. with the tip of tail 5 cm below the body. The bleeding timewas defined as the time required for the stream of blood to cease. Theinfusion of soluble P-selectin reduced bleeding time in hemophilia Amice (factor VIII-deficient mice).

[0269] As shown in FIG. 10, bleeding time was significantly decreasedfor hemophilia A mice treated with soluble P-selectin-Ig as compared tohemophilia A mice treated with human IgG1.

[0270] E. Activated Partial Thromboplastin Time (APTT)

[0271] Activated partial thromboplastin time (APTT) is a globalcoagulation screening test. It involves the intrinsic pathway ofcoagulation.

[0272] The effect on soluble P-selectin on activated partialthromboplastin time and plasma clotting time in factor VIII-deficientmice (hemophilia A mice) was determined as follows. Briefly, hemophiliaA mice were treated with 1.2 μg/g body weight P-selectin-Ig or humanIgG1. Mice were bled into ACD six hours after perfusion. Platelet poorplasma was prepared as described above. Activated partial thromboplastintime (APTT) was determined with APTT reagent and clotting was initiatedby addition of calcium ions. APTT and plasma clotting time are reducedin soluble P-selectin-Ig treated hemophilia A mice.

[0273] As shown in FIG. 14, APTT in soluble P-selectin-Ig treatedhemophilia A mice was shorter as compared to mice treated with humanIgG1 (p<0.0013, determined by unpaired t test). Recalcified clottingtime of plasma of hemophilia A mice treated with soluble P-selectin-Igwas significantly reduced (p<0.0058, determined by unpaired t test) ascompared to mice treated with control IgG1.

[0274] The foregoing Examples demonstrate the hemostatic activity ofsoluble P-selectin. The infusion of soluble P-selectin into a mouseinduces a procoagulant state in the animal. When such an animal iswounded, fibrin is deposited more rapidly at the site of the vesselinjury thus reducing leakage from the blood vessels. The plasma of theanimal infused with soluble P-selectin clots faster. Transgenic animalsexpressing higher levels of soluble P-selectin (ΔCT mice) also formfibrin more readily than wild-type animals and are protected fromexcessive leakage in hemorrhagic injury. In contrast, animals lackingall forms of P-selectin have an increased hemorrhagic response andslightly longer bleeding time than wild type. These data indicate thatthe level of soluble P-selectin is a predictor of coagulation potentialin a mammal.

[0275] Moreover, we have observed that infusion of soluble P-selectininto a mouse increases the numbers of microparticles containing tissuefactor in the blood. Similarly, transgenic mice expressing higher thannormal levels of soluble P-selectin have more tissue factor-containingmicroparticles in circulation. Infusion of soluble PSGL-1 (aligand/inhibitor of P-selectin) reduces the numbers of tissuefactor-containing microparticles and prolongs clotting time of theplasma in these mice. Thus, modulating P-selectin activity by, forexample, modulating levels of soluble P-selectin can either increase ordecrease hemostatic potential in a subject, and thus is useful for thediagnosis and treatment of hemostatic disorders.

Example 3 Soluble P-selectin Generates Microparticles in Human Blood

[0276] An in vitro system was developed to further demonstrate howsoluble P-selectin induces pro-coagulant activity. Generation ofmicroparticles after the addition of 15 μg/ml of human P-selectin-Igchimera or control human IgG1 was determined as described herein. Humanblood was collected in ACD. The blood samples from four donors, eachtreated separately, were incubated at 37° C. Samples were handled underaseptic conditions to avoid LPS contamination. The generation ofmicroparticles was analyzed by flow cytometry in platelet poor plasmadiluted in PBS. Forward scatter and sideward scatter plot was used forthe quadrant analysis to quantify the newly formed large procoagulatntmicroparticles. Tissue factor positive microparticles were analyzed byflow cytometry. The microparticles were stained with a FITC-conjugatedmouse anti-human tissue factor (American Diagnostica™).

[0277] As shown in FIG. 12A, after 6 hours incubation with solubleP-selectin, the numbers of procoagulant microparticles were increased by30% as compared to human IgG control (*=p<0.04).

[0278] As shown in FIG. 12B, the number of tissue factor positive evenswas significantly increased by incubation with soluble P-selectin-Ig in6 hours by 30% (*=p<0.05).

Example 4 Soluble P-selectin Shortens Whole Blood and Plasma ClottingTime in Human Blood

[0279] Whole blood recalcified clotting time and plasma recalcifiedclotting time in human blood after the addition of 15 μg/ml of humanP-selectin-Ig chimera or control human IgG1 was determined as follows.The human blood was collected in ACD. The blood samples from fourdonors, each treated separately, were incubated at 37° C. Samples werehandled under aseptic conditions to avoid LPS contamination. The wholeblood clotting time was measured in siliconized tubes in a SoloclotCoagulation and Platelet Analyzer (Sienco™).

[0280] As shown in FIG. 13A, the whole blood clotting time of humanblood incubated with soluble P-selectin was shortened by about 20% after2 hours (*=p<0.02) and by 60% after 8 hours of incubation (**=p<0.004)as compared to blood treated with IgG.

[0281] As shown in FIG. 13B, the plasma clotting time of the solubleP-selectin blood was shortened by 25% after 6 hours of incubation and by40% after 8 hours of incubation. (**p<0.004) as compared to control IgGand untreated plasma.

[0282] Equivalents

[0283] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed:
 1. A method for inducing hemostasis in a subject,comprising administering to said subject an inducer of P-selectinactivity, such that hemostasis occurs.
 2. The method of claim 1, whereinthe inducer of P-selectin activity increases the level of solubleP-selectin polypeptide in the plasma of the subject.
 3. The method ofclaim 2, wherein the inducer of P-selectin activity increases theproteolytic cleavage of P-selectin from a cell surface.
 4. The method ofclaim 2, wherein the inducer of P-selectin activity increases P-selectingene expression.
 5. The method of claim 1, wherein the inducer ofP-selectin activity binds to a P-selectin receptor or ligand and mimicsthe activity of a P-selectin polypeptide.
 6. The method of claim 5,wherein the inducer of P-selectin activity is an antibody to aP-selectin receptor or ligand.
 7. The method of claim 5, wherein theP-selectin ligand is PSGL-1.
 8. The method of claim 6, wherein theantibody is an antibody to PSGL-1.
 9. A method for inducing hemostasisin a subject, comprising administering to said subject a solubleP-selectin polypeptide, such that hemostasis occurs.
 10. A method forinducing hemostasis in a subject, comprising administering to saidsubject an isolated nucleic acid molecule comprising a nucleotidesequence which encodes a soluble P-selectin polypeptide, such thathemostasis occurs.
 11. A method for inducing hemostasis in a subject,comprising administering to said subject a recombinant cell expressingsoluble P-selectin polypeptide, such that hemostasis occurs.
 12. Amethod for treating or preventing a disorder associated withhypocoagulation in a subject, comprising administering to said subjectan inducer of P-selectin activity, such that the disorder associatedwith hypocoagulation is treated or prevented.
 13. The method of claim12, wherein said disorder is a hemorrhagic disorder.
 14. The method ofclaim 12, wherein said disorder is hemophilia.
 15. The method of claim12, wherein the inducer of P-selectin activity increases the level ofsoluble P-selectin polypeptide in the plasma of the subject.
 16. Amethod for treating or preventing a disorder associated withhypocoagulation in a subject, comprising administering to said subject asoluble P-selectin polypeptide.
 17. A method for treating avasculature-associated disease in a subject, comprising administering tosaid subject an inducer of P-selectin activity, such that thevasculature-associated disease is treated.
 18. The method of claim 17,wherein said vasculature-associated disease is a tumor.
 19. The methodof claim 18, wherein said subject is further treated with a moleculeeffective to induce a procoagulant state in tumor associatedvasculature.
 20. The method of claim 19, wherein said molecule comprisesa first binding region that binds to a component of a tumor cell ortumor associated vasculature, operatively linked to a coagulation factoror a second binding region that binds to a coagulation factor.
 21. Themethod of claim 20, wherein said first binding region comprises anantibody, or an antigen binding fragment thereof, that binds to VCAM-1,operatively linked to tissue factor.
 22. The method of claim 17, whereinthe inducer of P-selectin activity increases the level of solubleP-selectin polypeptide in the plasma of the subject.
 23. A method fortreating a vasculature-associated disease in a subject, comprisingadministering to said subject a soluble P-selectin polypeptide.
 24. Amethod for reducing hemostasis in a subject, comprising administering tosaid subject an inhibitor of P-selectin activity, such that procoagulantactivity is reduced.
 25. The method of claim 24, wherein the inhibitorof P-selectin activity decreases the level of soluble P-selectinpolypeptide in the plasma of the subject.
 26. The method of claim 25,wherein the inhibitor of P-selectin activity decreases the proteolyticcleavage of P-selectin from the cell surface.
 27. The method of claim26, wherein the inhibitor of P-selectin activity decreases P-selectingene expression.
 28. The method of claim 24, wherein the inhibitor ofP-selectin activity is an anti-P-selectin antibody.
 29. The method ofclaim 24, wherein the inhibitor of P-selectin activity is recombinantsoluble PSGL-1.
 30. A method for reducing hemostasis in a subject,comprising administering to said subject an isolated nucleic acidmolecule comprising a nucleotide sequence which is antisense to anucleotide sequence which encodes a P-selectin polypeptide, such thathemostasis is reduced.
 31. A method for treating or preventing athrombotic disorder in a subject, comprising administering to saidsubject an inhibitor of P-selectin activity, such that the thromboticdisorder is treated or prevented.
 32. The method of claim 31, whereinsaid disorder is arteriosclerosis.
 33. The method of claim 31, whereinsaid disorder is deep vein thrombosis.
 34. The method of claim 31,wherein said disorder is angina.
 35. The method of claim 31, whereinsaid thrombotic disorder is restenosis following medical intervention.36. The method of claim 31, wherein the inhibitor of P-selectin activitydecreases the level of soluble P-selectin polypeptide in the plasma ofthe subject.
 37. A method for modulating hemostatic potential in asubject, comprising modulating P-selectin activity in said subject. 38.The method of claim 37, wherein said modulating step comprisesadministering to the subject a modulator of P-selectin activity.
 39. Themethod of claim 38, wherein the modulator regulates the level of solubleP-selectin in the plasma of said subject.
 40. The method of claim 38,wherein the modulator is an inhibitor of P-selectin activity.
 41. Themethod of claim 38, wherein the modulator is an inducer of P-selectinactivity.
 42. A method for diagnosing a procoagulant state in a subject,comprising determining a P-selectin activity in a biological sample ofthe subject, wherein an increased P-selectin activity in the sampleindicates a procoagulant state in the subject.
 43. The method of claim42, which comprises providing a test sample of blood from a subject andcomparing the level of soluble P-selectin in the test sample to thelevel of soluble P-selectin in a control blood sample from a subjectwith normal hemostatic activity, wherein an increased level of solubleP-selectin in the test sample as compared to the control sample is anindication of a procoagulant state in the subject.
 44. A method ofidentifying a subject having a thrombotic disorder, or at risk fordeveloping a thrombotic disorder, comprising determining a P-selectinactivity in a biological sample of the subject, wherein an increasedP-selectin activity in the sample identifies a subject having athrombotic disorder, or at risk for developing a thrombotic disorder.45. The method of claim 44 comprising: a) contacting a sample of bloodobtained from said subject with a P-selectin binding substance; and b)detecting the presence of increased levels of soluble P-selectin in saidsample, thereby identifying a subject having a thrombotic disorder, orat risk for developing a thrombotic disorder.
 46. A method foridentifying a compound capable of modulating hemostasis, comprisingassaying the ability of the compound to modulate a P-selectin activity,thereby identifying a compound capable of modulating hemostasis.
 47. Themethod of claim 46, wherein the P-selectin activity is the expression ofsoluble P-selectin.
 48. A pharmaceutical composition for modulatinghemostasis comprising a compound identified according to the method ofclaim
 46. 49. A pharmaceutical composition for modulating hemostasiscontaining at least one compound which is a modulator of P-selectinactivity.